The White Sands pupfish (Cyprinodon tularosa), endemic to New Mexico in Southwestern North America, is of conservation concern due in part to invasive species, chemical pollution, and groundwater withdrawal. Herein, we developed a high quality draft reference genome and use it to provide biological insights into the evolution and conservation of C. tularosa. Specifically, we localized microsatellite markers previously used to demarcate Evolutionary Significant Units, evaluated the possibility of introgression into the C. tularosa genome, and compared genomic diversity among related species. The de novo assembly of PacBio Sequel II error-corrected reads resulted in a 1.08Gb draft genome with a contig N50 of 1.4Mb and 25,260 annotated protein coding genes, including 95% of the expected Actinopterigii conserved orthologs. Many of the previously described C. tularosa microsatellite markers fell within or near genes and exhibited a pattern of increased heterozygosity near genic areas compared to those in intergenic regions. Genetic distances between C. tularosa and the widespread invasive species C. variegatus, which diverged ~1.6-4.7 MYA, were 0.027 (nuclear) and 0.022 (mitochondrial). Nuclear alignments revealed putative tracts of introgression that merit further investigation. Genome-wide heterozygosity was markedly lower in C. tularosa compared to estimates from related species, likely because of smaller long-term effective population sizes constrained by their isolated and limited habitat. These population inferences, generated from our new genome assembly, provide insights into the long term and contemporary White Sands pupfish populations that are integral to future management efforts.