The Khapra beetle, Trogodermagranarium is one of the most common notorious stored grains pests in the tropical and subtropical regions of the world.Excessive and unplanned use of insecticides causes insects to developed resistance against many of them. It is greatly important to know the exact mechanism behind this resistance to successfully control this pest. Pakistan is an agricultural country that rely on agriculture for its economy facing persistent resistance problem and as a result lot of money, time and labour gone wasted each year. Therefore the main objective of the present study was to evaluate different detoxifying enzyme systems which are indicators of resistance. These indicator enzymes include esterases, glutathione-s-transferases and phosphatases as well as various metabolites and macromolecules. In current investigation, different populations of T. granariumtolerant to most commonly used pyrethroid, deltamethrin were collected from various godowns of the Punjab (Gujranwala, Okara and DG Khan). The possible role of esterases, glutathione-s transferases and phosphatases in tolerance to deltamethrin in T. granarium was evaluated. During this study an effort has been made to determine single cell DNA damages (if any) through Comet assay and possible association of different metabolites, metabolizing enzymes and macromolecules (soluble proteins, total lipids, total proteins, free amino acids, glucose, glycogen, trehalose, trehalsae, invertase and amylases) in development of tolerance in T. granarium. On the basis of LC50 of deltamethrin, the insecticide resistance gradation of various population are followed as Gujranwala >Okara> DG Khan > susceptible.Adult beetles of all populations were found to be most susceptible than other developmental stages. On the basis of LC50 values, different developmental stages of all populations are graded as 4th instar larvae > 6th instar larvae > adult beetles. In the present study, in 4thinstar and 6thinstar larvae and adult beetles, the various metabolites like soluble proteins, free amino acid, glucose contents along with carbohydrate metabolizing enzymes like, trehalase, amylase and invertase were significantly increased in all tolerantpopulations with reference to susceptible population. Based on increased levels of soluble proteins, free amino acid, glucose contents, trehalaseactivity, amylase activityand invertaseactivity, various tolerantpopulations can be graded as Gujranwala >Okara> DG Khan > susceptible.On the other hand, lipid contents, total proteins,glycogen contents and trehalose contents were decreased significantly in all tolerantpopulations. On the basis of decreased levels of lipid contents, total proteins,glycogen contents and trehalose contents, various tolerantpopulations can be graded as DG Khan >Okara> Gujranwala. The level of various esterases like total esterases (TE), choline esterases (ChE), acetylcholine esterases (AchE), aryl esterases (AE) and carboxyl esterases (CE) in 4thinstar and 6th instar larvae and adult beetles of tolerantpopulations viz., Gujranwala, Okara and DG Khan of T. granariumwas estimated and compared to susceptible population. Results showed that the level of TE, ChE, AChE, CE and AE activities were significantly increased in all tolerantpopulations as compared to susceptible population. Different developmental stages appear significantly more tolerant than adult beetles as 4th instar larvae was most tolerant than 6th instar larvae and 6th instar larvae was more tolerant than adult beetles significantly. These results showed that there is clear correlation between increased levels of esterases and tolerance in this stored grain insect pests. On the basis of increased levels of esterases activity in different developmental stages insect populations can be graded as Gujranwala >Okara> DG Khan > Susceptible. Among different development stages within all populations, a decreasing trend was found in the activities of esterases from 4th instar larvae to 6th instar larvae and adult beetles except 6th instar larvae of DG Khanpopulationwhich possessed higher TEactivity and 6th instar of Gujranwala populations whichpossessed higher AE activity than 4th instar larvae. Similarly adult beetles of Gujranwala population exhibited higher ChE activity than 6th and 4th instar larvae. The ChE activity of 4th instar larvae was less than 6th instar larvae in all less susceptible populations except Gujranwala. In Gujranwala population 4th instar larvae exhibited more ChE activity than 6th instar larvae and in DGKhan Population there is non significant difference among 4th instar and 6th instar larvae. Acid phosphatase, alkaline phosphatase and glutathione-s-transferaseactivity in three developmental stages (4thinstar and 6th instar larvae and adult beetles) of susceptible and three tolerantpopulations (Gujranwala, Okara and DG Khan) of T. granariumwere also assessed in present study. Acid phosphatase, alkaline phosphatase and glutathione-s transferaseactivity in all deltamethrintolerantpopulations was increased significantly as compared to susceptible population at P<0.05. On the basis of increased levels of these enzymes, different tolerantpopulations can be graded as Gujranwala >Okara> DG Khan.Withineach tolerant population among different developmental stages, acid phosphatase, alkaline phosphatase and glutathione-s-transferaseactivity was significantly different from each other at P<0.05. The 4th instar larvae exhibited less acid phosphataseand GSTactivity than that of 6th instar larvae and adult beetles in all tolerantpopulations and activity of adult beetles was less than 6th instar larvae in all tolerantpopulations as compared to susceptible population while 6th instar larvae and adult beetles possessed less alkaline phosphatase activity than that of 4th instar larvae in all populations and activity of adult beetles was less than 6th instar larvae in all tolerantpopulations. The comet parameters, namely olive tail moment (arbitrary units),head DNA(%), tail DNA (%) and tail length (μm) in the 4th instar larvae of susceptible and three deltamethrintolerantpopulations (Gujranwala, Okara and DG Khan) of T. granariumwerestudied. The 4th instar larvae of Gujranwala, Okara and DG Khan populationsshowed a significant decrease in head DNA as compared to susceptible population while increase in tail DNA (%), tail length (μm) and tail moment was exhibited by all tolerantpopulations respectively as compared to susceptible population. On the basis of increased levels of the comet parameters, different tolerantpopulations can be graded as Gujranwala >Okara> DG Khan.