The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is one of most destructive insect pests of agriculture and horticulture worldwide. It is a cryptic species complex, and biotypes of the complex have become serious pests in Pakistan because of their feeding and their ability to transmit cotton leaf curl virus (CLCuV). During 2007- 2008 and 2008-2009, the survey covered 40 cotton growing areas of the Punjab and Sindh provinces. Bemisia tabaci populations were sampled from cotton fields showing CLCuV infection. 100% prevalence was observed on the basis of typical begomovirus symptoms in cotton fields, both the years in Punjab and Sindh, Pakistan. The genetic diversity, biotype status of the Bemisia tabaci and its association with difference in Cotton Leaf Curl Disease incidence across both provinces were assessed by using random amplified polymorphic DNA-polymerase chain reaction and the mitochondrial cytochrome oxidase 1 (mtCO1) gene sequences. Phylogenetic analyses of mitochondrial cytochrome oxidase 1 sequences conducted by maximum-likelihood and maximum parsimony methods. The RAPD banding patterns showed considerable genetic variations among all 82 B.tabaci populations analyzed from each year. RAPD profiles generated 151 scorable amplification products, of which 77% were polymorphic in 2007 -2008 populations however 152 scorable amplification products were produced, of which 79% were polymorphic during 2008-2009 analysis. The study showed that the primer OPA-04 gave the unique and unambiguous DNA profiles that clearly distinguished biotype B from indigenous non B individuals. The primers gave polymorphic products of 150bp and 450bp with B biotype whereas 490bp and 1250bp with non B biotype. RAPD data clustered the B. tabaci samples on the basis of B and non B biotypes, regardless of the geographical origin of the populations. In both years analysis, 78 out of 80 Pakistani samples excluding the control populations fell in the non B cluster. The sub grouping between B.tabaci is diverse as Punjab populations set together with both Punjab and Sindh populations in this cluster. The B biotype populations formed an independent cluster contained B biotype reference population with two populations from Sindh. This shows that the presence of B biotype now termed Middle East Asia Minor 1 (MEAM 1) was restricted to a few locations of Sindh. This RAPD analysis showed that 96% of total B.tabaci samples studied are non B biotype (now termed Asia II 1), observed in cotton growing areas of Punjab and Sindh, Pakistan. These non B biotype B. tabaci are more diverse and indigenous to B biotype. It is estimated that genetic variations among non B populations increased by 2% over a year. Phylogenetic analyses of mtCO1 gene sequences congregated Pakistani B.tabaci with two genetic groups; Asia II 1(H, M, NA, non B biotypes) and MEAM 1 (B, B2 biotypes) in the year 2007-2008. However, the 2008-2009 samples fell into three distinctive clusters namely, Asia II 1, Asia II 5 and MEAM1. This phylogenetic inference revealed that the vast majority of the B.tabaci were Asia II 1 (non B biotype) and appeared well established in cotton growing regions of Punjab and Sindh province. This Asia II 1 cluster separated into two subgroups; subgroup A and subgroup B. The subgroup A, showed Pakistani populations shared close relationship with the Chinese, Indian and previously described Pakistani B.tabaci whereas the subgroup B clustered, Pakistani Bemisia populations with Nepal and Bangladesh populations. The high nucleotide identities (upto 99%) were observed in each group. MEAM1 was not detected in Punjab cotton growing areas by RAPD and phylogenetic analysis however identified from few locations of Sindh. Two populations from Pakpatan, Punjab and one from Khairpur, Sindh grouped with Asia II 5 (G biotype) with a high bootstrap support at 99%. The Asia II 5 is a new biotype observed in Pakistan and its presence is already reported in India and Bangladesh. This study is the first evidence of Asia II 5 presence in Punjab and Sindh, hence, the correlation between Asia II 5 and Cotton leaf curl disease (CLCuD) is yet unclear. The prevalence of Asia II 1 was sustained in these two year analysis of B.tabaci population from cotton. It seems that the distribution of this genetic group influence the distribution of CLCuD and plays a vital role in the spread of CLCuV all over Punjab and Sindh provinces, Pakistan. The phylogenetic study significantly supported the outcomes of RAPD analysis. The high genetic variations were observed in B.tabaci populations of cotton growing areas of Punjab and Sindh. The Asia II 1 (includes H, M, NA, non B biotypes) is prevalent in both provinces and associated with high incidence of CLCuD. The present study, gives the first indication of subsistence of Asia II 1 on cotton in Sindh and presence of Asia II 5 in Pakistan. This knowledge will contribute to the development of appropriate strategies with which to manage the disease in Pakistan.