Although the type specimen of California Grunion (Leuresthes tenuis) was described from a local fish market in 1860, this coastal marine fish was not seen in San Francisco again until 2001, when California Grunion were taken during trawl surveys in San Francisco Bay. In 2005, spawning California Grunion were confirmed on a sandy beach in San Francisco Bay for the first time. Fish were sampled from three locations to assess the Bay population's relationship to southern California Grunion populations in the traditional range of California Grunion, between Point Conception and northern Baja California, Mexico. Size differences, microsatellite loci, and mitochondrial sequences were examined to assess variation within and between these populations. Spawning adult California Grunion from San Francisco Bay are significantly smaller in length and mass from the southern California Grunion. Genetic data do not indicate substantial genetic divergence among the three populations, but there is evidence of a large number of single, unique haplotypes observed at all three sites. A large number of haplotypes suggests a large effective population size. We suggest the most likely scenario for the San Francisco population is a very recent colonization event following a northerly warm water pulse, possibly the El Niño–Southern Oscillation (ENSO) event of 1998–99, with the small population finding a thermal refuge in the Bay. Cold coastal water temperatures north of Point Conception may prevent colonization of the outer coast by northern populations of California Grunion. The differences in egg and adult size between the northern and southern populations are likely the result of phenotypic plasticity rather than genetic differences.