In the gift-giving spider Paratrechalea ornata, females mate multiple times and apparently may bias the paternity according to the nuptial gift content (nutritive or worthless). Paternity studies using microsatellite markers would, therefore, be ideal for studying male fertilization success for female offspring. We genotyped four microsatellites previously reported in one species of the genus using 43 individuals from four different localities in Uruguay. We describe the allelic and genotypic variation in all loci, and compare the findings to the reported variation for the genus. Except for one locus, all loci showed significant departure from the Hardy-Weinberg equilibrium due to heterozygote deficit. This could possibly be explained by the mixed nature of the sample and the effect of the population structure. These loci, and especially those that are highly hypervariable, are an important tool for paternity assessment and would allow a better understanding of the role of post-copulatory processes in P. ornata.