The genetic variability for a sample of 227 animals from three populations of Pantaneiro horses was estimated using data from 10 microsatellite loci. The number of alleles and the proportion of heterozygosity indicated high variability. A total of 91 alleles were found, with a significantly high mean number of alleles. The mean polymorphic information content was 0.7 and the paternity exclusion probability was 99.3%. The inbreeding coefficient (F(IS)) was low for the three populations: Ipiranga (F(IS) = 0.147), Nova Esperança (F(IS) = 0.094) and Promissão (F(IS) = 0.108). Genetic differentiation among all three populations was low (F(ST) = 0.008 to 0.064). Three methods were used to test for a recent bottleneck effect. The graphical method and the Wilcoxon test using the stepwise mutation model showed no bottleneck pattern for any of the populations. The test by two-phase mutation model showed genetic signatures of bottleneck for Ipiranga and Promissão. When we consider standard deviation value for Nova Esperança, the M-statistic detected a bottleneck pattern, but this result could be explained by a sample size effect. Therefore, there is no immediate cause for concern regarding loss of variation within the breed.