California vernal pool (VP) ecosystems support a diverse community of endemic plants that are threatened by multiple anthropogenic pressures, generating a need for molecular tools to quantify the extent and distribution of genetic variation in native populations. Here, we used RADseq to discover single nucleotide polymorphisms (SNPs) for a widespread VP endemic plant species, Lasthenia fremontii. We discovered nuclear-based SNPs using a RAD-tag library of 12 individuals from different VP complexes using SbfI, a restriction enzyme that does not cleave the chloroplast genome in Lasthenia. A total of 316,106 catalog loci were obtained across all twelve individuals in the library. Of these, 713 loci were polymorphic, yielding 3918 candidate SNPs. Next, we genotyped an additional 285 additional plants to validate and characterize 71 of the candidate SNPs. Of these, 44 were polymorphic among VP complexes. A preliminary analysis of the distribution of genetic variation using these loci revealed significant isolation-by-distance across the species’ geographic range. Weaker, but in some cases significant, genetic differentiation was detected among subpopulations from different pools within a single VP complex. Thus, in this study, RADseq allowed the discovery of SNP markers that can characterize patterns of genetic variation at multiple spatial scales in L. fremontii, which can be used to inform the conservation and mitigation of VP populations.