The California freshwater shrimp is a low dispersal species endemic to Northern California streams, where its habitat is naturally fragmented and has been further divided by human urbanization and agricultural activities. This species has been persisting for more than 200 million years, but in the last century, population decline resulted in its listing as an endangered species. A management and recovery plan was designed and the management units were designed based on drainage systems (watersheds) of the species and presence absence data. This analysis uses population genetic framework to empirically delineate gene pool boundary, estimate gene flow and effective population sizes. Single Nucleotide Polymorphism markers were developed de novo through ddRAD-seq methods suitable for non-model organisms, resulting in 357 unlinked loci scored with high confidence and little missing data. Evidence of strong genetic structure was observed among the sites sampled. Two hierarchical levels of differentiation were observed. The higher level containing four clusters (regional gene pools, FST between 0.40 – 0.72) isolated by low gene flow was associated with watershed limits. Local structure consisted of tributary streams within a watershed, which are connected through moderate gene flow (local gene pools, FST between 0.06 – 0.11). Within a stream, connectivity among sites was higher and no differentiation was observed. Estimates of current gene flow suggest that the watersheds are isolated from one another with limited to no gene flow in the last few generations. In addition to being isolated, these watersheds have small effective population sizes (4 – 250). Genetic diversity was lower (He ranged between 0.09 – 0.22) and variable across sampling sites and watersheds. These findings highlight the vulnerability of low dispersal freshwater shrimp to habitat fragmentation, and provide information for managers to increase connectivity between gene pools and effective population sizes.