ABSTRACT: BACKGROUND: Sulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been used in treatment of falciparum and vivax malaria in Nepal. Recently, resistance to both drugs have necessitated a change towards artemisinin combination therapy (ACT) against Plasmodium falciparum in highly endemic areas. However, SP is still used against P. falciparum infections in low endemic areas while CQ is used in suspected cases in areas with lack of diagnostic facilities. This study examines the prevalence of molecular markers of P. falciparum and Plasmodium vivax CQ and SP resistance to determine if high levels of in vivo resistance are reflected at molecular level as well. METHODS: Finger prick blood samples (n=189) were collected from malaria positive patients from two high endemic districts and analysed for single nucleotide polymorphisms (SNPs) in the resistance related genes of P. falciparum and P. vivax for CQ (Pfcrt, Pfmdr1, Pvmdr1) and SP (Pfdhfr, Pfdhps, Pvdhfr), using various PCR-based methods. RESULTS AND DISCUSSION: Positive P. vivax and P. falciparum infections were identified by PCR in 92 and 41 samples respectively. However, some of these were negative in subsequent PCRs. Based on a few P. falciparum samples, the molecular level of CQ resistance in P. falciparum was high since nearly all parasites had the Pfcrt mutant haplotypes CVIET (55%) or SVMNT (42%), though frequency of Pfmdr1 wild type haplotype was relatively low (35%). Molecular levels of SP resistance in P. falciparum was found to be high. Most prevalent Pfdhfr haplotype was double mutant CNRNI (91%), while frequency of Pfdhps double mutant SGEAA and AGEAA were 38% and 33% respectively. Combined, the frequency of quadruple mutations (CNRNI-SGEAA/AGEAA) was 63%. Based on P. vivax samples, low CQ and SP resistance were most likely due to low prevalence of Pvmdr1 Y976F mutation (5%) and absence of triple/quadruple mutations in Pvdhfr. CONCLUSIONS: Based on the limited number of samples, prevalence of CQ and SP resistance at molecular levels in the population in the study area were determined as high in P. falciparum and low in P. vivax. Therefore, CQ could still be used in the treatment of P. vivax infections, but this remains to be tested in vivo, while the change to ACT for P. falciparum seems justified.