Levels of drug resistance of Plasmodium falciparum strains against antimalarials have increased in Laos. In several studies, chloroquine (CQ) resistance has been associated with point mutations in the Pfcrt and pfmdr genes, and sulphadoxine/pyrimethamine (S/P) resistance with point mutations in the genes of dihydrofolate reductase ( DHFR) and dihydropteroate synthetase ( DHPS). We combined a study of these molecular markers with an in vivo antimalarial drug sensitivity study in Attapeu province in the south of Lao PDR. We treated 100 patients with either CQ, S/P or a combination of both. In the CQ group, Pfcrt mutations showed a very high sensitivity (100%) but a low specificity (12.5%) to predict resistance. The combination of mutations in the Pfcrt and pfmdr genes was highly specific and had a positive predictive value of 100%. Mutations in the DHPS gene showed a high correlation with the development of resistance. The prevalence of mutations in the DHFR gene, especially codon 108 Asn, was predictive with high sensitivity ( 100%) but low specificity. Isolates derived from patients treated with a combination of both drugs showed a high correlation between the mutation in codon 437 of DHPS gene and in vivo-resistance ( odds ratio 16.00, CI). The study provides evidence for the existence of antimalarial drug resistance in the south of Lao PDR, and offers a molecular method to predict resistance.