The first report of Plasmodium falciparum chloroquine (CQ) resistance (CQR) in Papua New Guinea (PNG) appeared in 1974. Although the current prevalence of CQR-associated parasite gene polymorphisms has been documented for some regions, the spatial and temporal relationships that characterize CQ-resistant parasites in PNG are unknown. Insight into the evolution of CQ-resistant parasites could be provided by evaluating genetic markers in parasite populations. We compared pfcrt and pfmdr1 polymorphisms and flanking microsatellite (MS) polymorphisms between P. falciparum-infected placental tissue (early 1980s) and blood (late 1990s) samples collected throughout PNG. Consistent with the results of recent studies, pfcrt-SVMNT and pfmdr1-86Y were the only CQR-associated alleles observed in the placental tissue samples, and they were observed together in 79% of the samples. Results of analysis of MS flanking pfcrt (approximately 40 kb) suggested that there was less diversity in the samples collected during the 1980s than in those collected during the 1990s and that the 1990s parasites were significantly differentiated from the 1980s parasites. On the other hand, for MS flanking pfmdr1 (approximately 5 kb) and for 1 putatively neutral locus, diversity levels were similar, and the 2 parasite populations were not significantly differentiated. These results suggest that selection for CQR was operating on the pfcrt-SVMNT allele during the early 1980s. Thus, archival samples can provide novel insight into the dynamics of CQR.