Four farmed stocks and four wild populations of Nile tilapia (Oreochromis niloticus), which was first introduced to Madagascar sixty years ago, were assayed for genetic variation at nine microsatellite loci to determine levels of genetic diversity within populations and genetic relationships between them. Allelic diversity overlapped with that found in previously sampled populations elsewhere in Africa. There was no evidence of deviations from allele frequencies expected under conditions of Hardy-Weinberg equilibrium or of inbreeding in studied populations. Three distinct clusters of genotypes provided evidence of three separate introductions (from Egypt and Mauritius in 1956, and from Japan in 2011), and the occurrence of genotypes from more than one cluster within a single population provided evidence of their mixing. There were significant differences between populations which were not from the same environment (wild or farmed) or were not geographically related. Wild populations may be a valuable resource to support further development of farmed stocks from the perspective of genetic diversity.