The identification of the cryptic diploid-tetraploid Gray Treefrogs, Hyla chrysoscelis and H. versicolor, from preserved specimens is problematic using conventional methods, such as karyotyping. Nucleolar number has been used as an index of ploidy for this purpose, based on the assumption of a single nucleolus per chromosome set. However, cytogenetic studies of nucleolar organizer regions (NORs) have shown that putative secondary NORs occur in some populations of the diploid, H. chrysoscelis. This study demonstrates that secondary NORs in the diploid genome are associated with additional nucleoli in interphase cell nuclei. The number of nucleoli is proportional to the total number of primary and secondary NORs, not ploidy level, and, therefore, is not a valid character for identification of specimens from populations having secondary NORs. Because little is known about the frequency of secondary NORs in most H. chrysoscelis populations, nucleolar number should be avoided as a primary means of species identification.