A geographically isolated set of southern localities of the formerly monotypic goby genus Eucyclogobius is known to be reciprocally monophyletic and substantially divergent in mitochondrial sequence and nuclear microsatellite-based phylogenies relative to populations to the north along the California coast. To clarify taxonomic and conservation status, we conducted a suite of analyses on a comprehensive set of morphological counts and measures from across the range of Eucyclogobius and describe the southern populations as a new species, the Southern Tidewater Goby, Eucyclogobius kristinae, now separate from the Northern Tidewater Goby Eucyclogobius newberryi (Girard 1856). In addition to molecular distinction, adults of E. kristinae are diagnosed by: 1) loss of the anterior supratemporal lateral-line canals resulting in higher neuromast counts, 2) lower pectoral and branched caudal ray counts, and 3) sets of measurements identified via discriminant analysis. These differences suggest ecological distinction of the two species. Previous studies estimated lineage separation at 2–4 million years ago, and mitochondrial sequence divergence exceeds that of other recognized fish species. Fish from Santa Monica Artesian Springs (Los Angeles County) northward belong to E. newberryi; those from Aliso Creek (Orange County) southward constitute E. kristinae. The lagoonal habitat of Eucyclogobius has been diminished or degraded, leading to special conservation status at state and federal levels beginning in 1980. Habitat of the newly described species has been impacted by a range of anthropogenic activities, including the conversion of closing lagoons to open tidal systems in the name of restoration. In the last 30 years, E. kristinae has only been observed in nine intermittently occupied lagoonal systems in northern San Diego County; it currently persists in only three sites. Thus, the new species is in imminent danger of extinction and will require ongoing active management.