BACKGROUND The Plasmodium falciparum apical membrane antigen-1 (PfAMA1) is considered as an ideal vaccine candidate for malaria control due to its high level of immunogenicity and essential role in parasite survival. Among the three domains of PfAMA1 protein, hyper-variable region (HVR) of domain I is the most immunogenic. The present study was conducted to evaluate the extent of genetic diversity across HVR domain I of the pfama1 gene in P. falciparum isolates from Hazara division of Pakistan. METHODS The HVR domain I of the pfama1 was amplified and sequenced from 20 P. falciparum positive cases from Hazara division of Pakistan. The sequences were analysed in context of global population data of P. falciparum from nine malaria endemic countries. The DNA sequence reads quality assessment, reads assembling, sequences alignment/phylogenetic and population genetic analyses were performed using Staden, Lasergene v. 7.1, MEGA7 and DnaSP v.5 software packages respectively. RESULTS Total 14 mutations were found in Pakistani isolates with 12 parsimony informative sites. During comparison with global isolates, a novel non-synonymous mutation (Y240F) was found specifically in a single Pakistani sample with 5% frequency. The less number of mutations, haplotypes, recombination and low pairwise nucleotide differences revealed tightly linked uniform genetic structure with low genetic diversity at HVR domain I of pfama1 among P. falciparum isolates from Hazara region of Pakistan. This uniform genetic structure may be shaped across Pakistani P. falciparum isolates by bottleneck or natural selection events. CONCLUSIONS The Pakistani P. falciparum isolates were found to maintain a distinct genetic pattern at HVR pfama1 with some extent of genetic relationship with geographically close Myanmar and Indian samples. However, the exact pattern of gene flow and demographic events may infer from whole genome sequence data with large sample size of P. falciparum collected from broad area of Pakistan.