Surveillance for Plasmodium falciparum drug resistance mutations is becoming an established tool for assessing antimalarial treatment effectiveness. We used an extended version of a high-throughput post-PCR multiplexed ligase detection reaction fluorescent microsphere assay (LDR-FMA) to detect single-nucleotide P. falciparum drug resistance polymorphisms in 402 isolates from children in Papua New Guinea (PNG) participating in an antimalarial treatment trial. There was a fixation of P. falciparum crt (pfcrt) K76T, pfdhfr C59R and S108N, and pfmdr1 mutations (92%, 93%, 95%, and 91%, respectively). Multiple mutations were frequent. Eighty-eight percent of isolates possessed a quintuple mutation (underlined), SVMNT, NRNI, KAA, and YYSND, in codons 72 to 76 for pfcrt; 51, 59, 108, and 164 for pfdhfr; 540, 581, and 613 for pfdhps; and 86, 184, 1034, 1042, and 1246 for pfmdr1, and four of these carried the K540E pfdhps allele. The pfmdr1 D1246Y mutation was associated with PCR-corrected day 42 in vivo treatment failure in children allocated piperaquine-dihydroartemisinin (P = 0.004). Although the pfmdr1 NFSDD haplotype was found in only four isolates, it has been associated with artemether-lumefantrine treatment failure in Africa. LDR-FMA allows the large-scale assessment of resistance-associated single-nucleotide polymorphisms (SNPs). Our findings reflect previous heavy 4-aminoquinoline/sulfadoxine-pyrimethamine use in PNG. Since artemether-lumefantrine and piperaquine-dihydroartemisinin will become first- and second-line treatments, respectively, the monitoring of pfmdr1 SNPs appears to be a high priority.