The increasing use of sulfadoxine/pyrimethamine (SP) for treatment of chloroquine-resistant Plasmodium falciparum has resulted in increased exposure of Plasmodium vivax parasites in areas where both species co-exist. In this study, the extent of mutations/haplotypes in pvdhfr and pvdhps was examined using PCR-RFLP methods in 427 P. vivax isolates in Iran after 4 years of introducing SP as the first-line anti-malarial drug in Iran. Mutations were detected in three codons of pvdhfr (F57L, S58R and S117N) and in one of pvdhps (A383G) and the majority of isolates had double mutations (58R/117N, 45.4%). In addition, the frequency of 57L mutation was detected in 8.2% of P. vivax isolates. This frequency was significantly increased when compared with a similar study on P. vivax isolates in 2005 (X(2) test, P<0.0001). Moreover, there was an increase in the frequency of single nucleotide polymorphisms at position 383G in pvdhps (0-2.6%) was found. Furthermore, the number of haplotypes increased from 6 to 12 in the study areas during 2006-2010. Interestingly, when combining the two loci, the frequency of parasites carrying pvdhfr/pvdhps pure mutations (L(57)R(58)/G(383), R(58)N(117)/G(383)) increased from 0% in 2006 to 2.1% in 2010. In conclusion, the present results suggest that SP could be effective in treatment against the erythrocytic stages of vivax malaria in Iran; however, the increased frequency of mutant haplotypes in Iran since 2006 is worrying and indicates the emergence of drug-tolerant/resistant P. vivax isolates in Iran in near future.