Association between sensitivity to house dust mite (HDM) allergens and allergic disorders is well known. Modern housing, genetic traits and environmental pollution are the major causes of increased prevalence of HDM allergy. The aim of this study was to estimate the epidemiological status of HDM allergy in Pothwar region. Samples of house dust were investigated to identify the prevalent HDM species in the house environment and their allergen levels. Molecular characterization of group 1 allergens and their polymorphism in prevalent HDM species was also undertaken. The present study found, that, an estimated 81.74% of patients were sensitized to HDM allergens (Der p1 and Der f1), 87% patients were poly-sensitized, wherein patients showing sensitization to pollen and food allergens were 53% and 38% respectively. Percentage of HDM sensitization was highly significant compared to the other two allergy groups (p=0.002357). A significant gender bias was observed, with percentage of male patients (56.11%) greater than female patients (43.89%). Results revealed D. farinae and D. pteronyssinus as the predominant acrofauna. Out of total examined mites, 60.89% were identified as D. farinae, followed by D. pteronyssinus (29%). Besides these two species of allergy causing family of Pyroglyphids, 11% mites were non-allergenic i.e. Cheyletidae and Oribatidae. Comparison of mite counts from randomly selected houses and patients’ houses showed analogous counts of D. farinae and D. pteronyssinus. 87.35% random houses and 87.11% patients’ houses showed positive mite infestation. Mean D. farinae counts/gram of dust, in random samples were 235.36±7.93 (mean ± SEM) compared to 274.74±10.78 from patients’ homes. Similarly, mean D. pteronyssinus counts from random houses compared to patients’ houses were 115.04±4.57 and 124.58±5.76 respectively. Seasonal variation in mite counts was significant, with highest mite counts observed during monsoon season when % RH and ambient air temperatures are most suitable for mite proliferation. Allergen levels in 81.2% dust samples tested for Der f 1 were above 0.5ng/ml Limit of detection (LOD), where 57.6% dust samples had more than10μg/g dust allergen load. Conversely 69.4% of samples above were above LOD for Der p 1 and 20% had Der p 1 allergen levels greater than10μg/g dust. Mean Der f 1 (12.03±0.86μg/g) burden was significantly higher (p<0.0001) than mean Der p1 (6.06±0.73 μg/g). A moderate correlation (R2=0.6) between mite counts and their allergen levels was observed. Both Der f 1 and Der p 1 allergen levels were significantly high (p<0.0001) during monsoon and autumn compared to the remaining seasons of the year. Study of group 1 allergen polymorphism revealed two novel introns at nucleotide position (nt pos) 87 and 291 in both species, and the absence of intron 3 in Der p 1. Thirteen silent and one novel non-synonymous mutation: Tryptophan (W197) to Arginine (R197) were detected in D. farinae. Two haplotypes of Der f 1 gene were identified, haplotype 1 (63%) was more frequent than haplotype 2 (18%). In Der p 1, a silent mutation at nt (aa) position 1011(149) and four non-synonymous mutations at positions 589(50), 935(124), 971(136) and 1268(215) were observed. These mutations were reported from many other geographic regions, suggesting that polymorphism in Der p 1 gene is panmictic. As an outcome of this research, a better awareness (with relevant data) about the epidemiological status of House Dust Mite (HDM) allergy in Pothwar region has been established. Presence of Pyroglyphid mite species (D. farinae and D. pteronyssinus only) and the absence of other species from the family Pyroglyphidae are reported for the first time. The extent of polymorphism in both genes was substantially lower than that reported previously (0.10-0.16% vs 0.31-0.49%), indicating the need for careful evaluation of potential polymerase errors in studies utilizing RT-PCR.