In total, 129 Plasmodium vivax isolates from different geographical areas in India were analysed for point mutations in the P. vivax dihydrofolate reductase gene that were associated with pyrimethamine resistance. A gradual increase in the frequency of mutant genotypes was observed from north to south (p < 0.0001). In the northern region (Delhi, Panna and Nadiad), the wild-type genotype was most prevalent, while the mutant genotype predominated in the coastal regions of southern India (Navi Mumbai, Goa and Chennai). Isolates from the Car-Nicobar islands showed only mutant genotypes. The differential geographical pattern of mutations may be associated with the transmission pattern.