We evaluated the influence of pfmdr1 mutations and copy number on in vitro artemether and lumefantrine sensitivity in 101 laboratory and adapted Thai isolates of Plasmodium falciparum. Approximately one-fourth of these isolates exhibited reduced lumefantrine susceptibility. We found that both mutations and amplification of the pfmdr1 gene influenced in vitro artemether and lumefantrine sensitivity. Using multivariate analysis, 184F or 1042N alleles and a copy number of ≥ 4 were identified as the independent markers for decreased lumefantrine susceptibility. Separate analysis also indicated that parasites from different geographical areas were influenced by different genetic markers.