Understanding genetic structure and diversity among remnant populations of rare species can inform conservation and recovery actions. We used a population genetic framework to spatially delineate gene pools and estimate gene flow and effective population sizes for the endangered California Freshwater Shrimp Syncaris pacifica. Tissues of 101 individuals were collected from 11 sites in 5 watersheds, using non-lethal tissue sampling. Single Nucleotide Polymorphism markers were developed de novo using ddRAD-seq methods, resulting in 433 unlinked loci scored with high confidence and low missing data. We found evidence for strong genetic structure across the species range. Two hierarchical levels of significant differentiation were observed: (i) five clusters (regional gene pools, F_ST = 0.38–0.75) isolated by low gene flow were associated with watershed limits and (ii) modest local structure among tributaries within a watershed that are not connected through direct downstream flow (local gene pools, F_ST = 0.06–0.10). Sampling sites connected with direct upstream-to-downstream water flow were not differentiated. Our analyses suggest that regional watersheds are isolated from one another, with very limited (possibly no) gene flow over recent generations. This isolation is paired with small effective population sizes across regional gene pools (N_e = 62.4–147.1). Genetic diversity was variable across sites and watersheds (H_e = 0.09–0.22). Those with the highest diversity may have been refugia and are now potential sources of genetic diversity for other populations. These findings highlight which portions of the species range may be most vulnerable to future habitat fragmentation and provide management consideration for maintaining local effective population sizes and genetic connectivity.