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<p>Abstract</p> <p>Background</p> <p>Polymorphism in the <it>pfcrt </it>gene underlies <it>Plasmodium falciparum </it>chloroquine resistance (CQR), as sensitive strains consistently carry lysine (K), while CQR strains carry threonine (T) at the codon 76. Previous studies have shown that microsatellite (MS) haplotype variation can be used to study the evolution of CQR polymorphism and to characterize intra- and inter-population dispersal of CQR in Papua New Guinea (PNG).</p> <p>Methods</p> <p>Here, following identification of new polymorphic MS in introns 2 and 3 within the <it>pfcrt </it>gene (msint2 and msint3, respectively), locus-by-locus and haplotype heterozygosity (<it>H</it>) analyses were performed to determine the distribution of this intronic polymorphism among <it>pfcrt </it>chloroquine-sensitive and CQR alleles.</p> <p>Results</p> <p>For MS flanking the <it>pfcrt </it>CQR allele, <it>H </it>ranged from 0.07 (B5M77, -18 kb) to 0.094 (9B12, +2 kb) suggesting that CQ selection pressure was responsible for strong homogenisation of this gene locus. In a survey of 206 <it>pfcrt</it>-SVMNT allele-containing field samples from malaria-endemic regions of PNG, <it>H </it>for msint2 was 0.201. This observation suggests that <it>pfcrt </it>msint2 exhibits a higher level of diversity than what is expected from the analyses of <it>pfcrt </it>flanking MS. Further analyses showed that one of the three haplotypes present in the early 1980's samples has become the predominant haplotype (frequency = 0.901) in CQR parasite populations collected after 1995 from three PNG sites, when CQR had spread throughout malaria-endemic regions of PNG. Apparent localized diversification of <it>pfcrt </it>haplotypes at each site was also observed among samples collected after 1995, where minor CQR-associated haplotypes were found to be unique to each site.</p> <p>Conclusion</p> <p>In this study, a higher level of diversity at MS loci within the <it>pfcrt </it>gene was observed when compared with the level of diversity at <it>pfcrt </it>flanking MS. While <it>pfcrt </it>(K76T) and its immediate flanking region indicate homogenisation in PNG CQR parasite populations, <it>pfcrt </it>intronic MS variation provides evidence that the locus is still evolving. Further studies are needed to determine whether these intronic MS introduce the underlying genetic mechanisms that may generate <it>pfcrt </it>allelic diversity.</p>