Because of its geo strategic position at the crossroad of Asia, Pakistan has gained crucial importance of playing its pivotal role in subsequent human migratory events, both prehistoric and historic. This human movement became possible through an ancient overland network of trails called “The Silk Route” linking Asia Minor, Middle East China, Central Asia, and Southeast Asia. The present study was designed to investigate the control region in 500 unrelated individuals of 22 ethnic groups of the Pakistani population. In order to discourse the genetic diversity, affiliations and origin of castes of Punjab and tribes of Baluchistan, KPK and Sindh. The study revealed high genetic diversity in Pakistani population which is comparable to the other Central Asian, Southeast Asian and European populations. For this purpose Sequence analysis scrutinized, 412 haplotypes, defined by a particular set of nucleotides were found (ignoring the C insertions around position 309 and 315) in 22 ethnic groups. In spite of that 65% sequences were observed once, 11 %, twice, 8 % thrice, 5 % four time and 2.2 % five times. The most common South Asian haplotypes are observed, M 46 %, N 7 %, and R 13%, while West Eurasian haplotypes are U 18%, H 5%, J 4%, W 3% and T 2% in 22 ethnic groups. The mean number of pairwise differences were observed 5.2 ± 2.8 to 12.5 ± 6.2. A random match probability between two unrelated individuals was found between 0.01 to 0.06 %. While genetic diversity was found to be 0.991 to 0.999, with nucleotide diversity ranging from 0.0089 to 0.0142 for the whole control region in Pakistani population. The configuration of genetic variation and heterogeneity further unveiled through Multidimensional Scaling and phylogenetic analysis. The results revealed that Pakistani ethnic groups are the composite mosaic of West Eurasian ancestry of numerous geographic origin. They received substantial gene flow during different invasive movements and have a high element of the Western provenance.