Determining the genetic diversity and differentiation among populations is a critical element of conservation biology, but for many aquatic, data-deficient species with small population sizes, this is not possible. Closely related species may therefore provide a model. For the first time, using over 4000 single-nucleotide polymorphism loci, we characterise the population genetic diversity and structure of one of the world’s rarest marine fish, the spotted handfish (Brachionichthys hirsutus), a species which is also a member of the most threatened marine bony fish family (Brachionichthyidae). Fin clips were taken from 170 live spotted handfish across seven disjunct sites within the only known estuary (in Tasmania, Australia) where multiple populations of the species are found. Spatially discrete populations clustered into three genetic groupings and a significant variance in allele frequencies among populations (overall F_ST = 0.043), even at the small scale of the estuary, was observed. Furthermore, low contemporary migration rate estimates suggest low genetic homogeneity between locations. Because of the low genetic connectivity, population clusters of spotted handfish within the estuary should be considered as separate conservation management units. This insight should be considered for management and conservation strategies of other data-deficient and threatened species in the family.