Parental investment as manifested through extended parental care of young presumably enhances the reproductive success of the custodial parent. In the Tessellated Darter (Etheostoma olmstedi), the primary caregivers are breeding males on the nest. However, prior field observations on nesting darters seem suggestive of behaviors that are more difficult to interpret evolutionarily. These include tending clutches that may have been fertilized by other males and appropriating nests from smaller courting males. To address such possibilities genetically, we assayed six microsatellite loci in 16 nest-tending males and the embryos from their associated clutches. In most cases, a guardian male had sired nearly all of the embryos in his nest. However, in one nest, a guardian male had been cuckolded, and in two other nests, an attendant male guarded embryos that were not his own presumably resulting from nest takeovers. From direct genotypic counts, a mean of at least 3.2 mothers contributed to the progeny in a nest, and computer simulations suggest that the true maternal number may be substantially higher.