Major histocompatibility complex (MHC) class II molecules have an important role in vertebrate adaptive immunity, being responsible for recognising, binding and presenting specific antigenic peptides to T lymphocytes. We have studied the MHC class II DQB and DRB exon 2 genes of the Australian sea lion (Neophoca cinerea), an endangered pinniped species that experiences high pup mortality. Following characterisation of N. cinerea DQB and DRB by molecular cloning, and evaluation of diversity in pups across two colonies using variant screening (n = 47), three DQB alleles and ten DRB variants (including one pseudogene allele) were identified. The higher diversity at DRB relative to DQB is consistent with other studies in marine mammals. Despite overall lower MHC class II allelic diversity relative to some other pinniped species, we observed similar levels of nucleotide diversity and selection in N. cinerea. In addition, we provide support for recent divergence of MHC class II alleles. The characterisation of MHC class II diversity in the Australian sea lion establishes a baseline for further investigation of associations with disease, including endemic hookworm infection, and contributes to the conservation management of this species.