Relationships between mean individual fitness and genetic heterozygosity remain controversial because multiple studies over many years have yielded inconsistent results. However, molecular measurements of genetic variation have mostly been based on allozyme markers, which may have substantial limitations due to their relative insensitivity and occasionally also to the effects of natural selection. We investigated variation in the number of tandem dinucleotide repeats at eight polymorphic microsatellite loci in 38 natterjack toad, Bufo calamita, populations that varied in census size, degree of isolation and distance from the distributional range edge. We also measured fitness attributes, notably larval survival and growth rates, under standardized conditions using samples from six of these populations. The results indicated that larval growth rates were positively and strongly correlated with expected heterozygosity (He) across the microsatellite loci. He was in turn highest in large populations with minimal isolation, but low near the biogeographical range edge irrespective of population size. Larvae from the smallest and most isolated natterjack population exhibited particularly low fitness and heterozygosity. Our results suggest that microsatellite loci may provide a valuable new approach in studies of links between fitness and heterozygosity.