The genus Leuresthes displays reproductive behavior unique among marine fish in which mature adults synchronously emerge completely out of the water to spawn on beach land. A limited number of sandy beaches, which are suitable for these spawning events, are present in discontinuous locations along the geographic range of the species, potentially limiting gene flow and the degree of genetic homogeneity between intraspecific populations. Here, we tested for molecular genetic differentiation between 363 individuals, representing 3 populations of California grunion, Leuresthes tenuis, by employing 2 mitochondrial and 4 nuclear DNA markers. We include temporally diverse sampling to evaluate contemporary and temporal divergence, and we also analyze 28 individuals from one population of Gulf grunion (restricted to the Gulf of California), Leuresthes sardina, at the same markers to evaluate the molecular evidence for their separate species distinction. We find no significant differences between temporal samples, but small significant differences among all populations of L. tenuis, and unequivocal support for the separate species distinction of L. sardina. Genetic data suggest that the Monterey Bay population of L. tenuis near the species’ most northern range likely represents a relatively recent colonization event from populations along the species’ more traditional range south of Point Conception, California. We conclude that both the topographic features of the California and Baja California coastlines and the grunions’ unique reproductive behavior have influenced the genetic structure of the populations.