The federally endangered Cape Sable seaside sparrow (Ammospiza maritima mirabilis) is endemic to the Greater Everglades ecosystem in southern Florida, inhabiting fragmented marl prairies in six individual subpopulations. The subspecies is threatened by loss of breeding habitat from fire and water management. Genetic information is severely limited for the subspecies but could help inform decisions regarding subpopulation protections and potential translocations for genetic rescue. To provide genetic data and inform management efforts, feather samples were collected across five subpopulations (designated A–E) and protocols were tested to optimize DNA extraction yields. We assessed four mitochondrial DNA markers (N = 36–69) and 12 nuclear microsatellite loci (N = 55) in 108 sparrows. Mitochondrial DNA sequences revealed low haplotype diversity, with NADH dehydrogenase-2 haplotypes matching to most other extant subspecies and to the Atlantic coast subspecies. Nuclear diversity was low compared to other subspecies, but similar across subpopulations. Samples grouped as one population when analyzed by Principal Component Analysis, Bayesian modelling and genetic distance metrics. Limited genetic emigration was detected from one putative migrant. Relatedness was significantly different for sparrows in the most geographically distant subpopulation (A), likely reflecting high self-recruitment and natal site fidelity (P = 0.003). The low to moderate effective population size (N_E = 202.4; N_E:N_C = 0.06) and generation time estimates indicated that unique genetic variation could be lost quickly during stochastic events. The sample sizes were limited, which reduced the power to comprehensively address recent population size reductions and any subsequent loss of genetic diversity.