Genetic diversity at the Major Histocompatibility Complex (MHC) is particularly important for species viability as it allows populations to respond to emerging pathogens and infectious disease. Patterns of variation at this gene complex serve as a useful complement to information obtained from neutral loci for planning management and conservation strategies that seek to ensure the adaptive potential of at-risk species. In this study, we investigated patterns of genetic variation at exon 2 of the MHC class II gene in the critically endangered Fundulus julisia (Barrens Topminnow). This species has undergone dramatic declines over the last 30 years, leading to its recent proposal for federal protection under the Endangered Species Act. Patterns of nucleotide substitution and phylogenetic analyses revealing trans-species polymorphisms suggest that this locus has been of adaptive importance in the history of this species. Despite recent population declines and documented population bottlenecks, measures of genetic diversity were high in comparison to patterns observed at putatively neutral microsatellite and mitochondrial markers. Results from this study are discussed in the context of recovery plans for the Barrens Topminnow and lend support to the previous designation of evolutionary significant units and management units based on neutral markers.