Litoria spenceri is a critically endangered frog species found in several population clusters within Victoria and New South Wales, Australia. Biobanking of cell cultures obtained from toe clippings of adults originating from Southern, Northern and Central Site locations, as well as Northern x Central Site hybrid tadpole crosses was performed. Analysis of biobanked cells demonstrates a 2n = 26 karyotype and chromosomal morphology characteristic of the Litoria genus. A potential nucleolar organiser region (NOR) on chromosome 9 demonstrates similar designation to L. pearsoniana and L. phyllochroa of the same phylogenetic subgroup. A second potential novel NOR was also located on the long arm of chromosome 11, and only within the Central Site population. This Central Site apparent NOR is inheritable to Northern x Central Site tadpole hybrids in the heterozygous state and appears to be associated with a metacentric to submetacentric morphological transformation of the Northern Site inherited matched chromosome of that pair. This potential NOR represents an important genetic marker for distinguishing subpopulations. These data demonstrate the importance of prospectively establishing biobanks containing genetically characterized cells so that effective markers of specific subpopulations can be identified and used to help increase the effectiveness of animal husbandry programs.