Here we examined whether the recent dramatic decline in malaria transmission in Sri Lanka led to a major bottleneck in the local Plasmodium vivax population, with a substantial decrease in the effective population size. To this end, we typed 14 highly polymorphic microsatellite markers in 185 P. vivax patient isolates collected from 13 districts in Sri Lanka over a period of 5 years (2003–2007). Overall, we found a high degree of polymorphism, with 184 unique haplotypes (12–46 alleles per locus) and average genetic diversity (expected heterozygosity) of 0·8744. Almost 69% (n = 127) isolates had multiple-clone infections (MCI). Significant spatial and temporal differentiation (F
= 0·04–0·25; P⩽0·0009) between populations was observed. The effective population size was relatively high but showed a decline from 2003–4 to 2006–7 periods (estimated as 45 661 to 22 896 or 10 513 to 7057, depending on the underlying model used). We used three approaches – namely, mode-shift in allele frequency distribution, detection of heterozygote excess and the M-ratio statistics – to test for evidence of a recent population bottleneck but only the low values of M-ratio statistics (ranging between 0·15–0·33, mean 0·26) were suggestive of such a bottleneck. The persistence of high genetic diversity and high proportion of MCI, with little change in effective population size, despite the collapse in demographic population size of P. vivax in Sri Lanka indicates the importance of maintaining stringent control and surveillance measures to prevent resurgence.