Significant differences in the length–frequency composition of catch samples, mass–length relationships and length-at-maturity found between Heterodontus portusjacksoni from western and far-eastern Victoria, suggest the presence of at least two separate breeding populations off south-eastern Australia, with some mixing between these regions. In each region females attain a larger size than males, and collectively those sharks in western Victoria are smaller, present higher mass-at-length, and lower length-at-maturity than those in far-eastern Victoria. These regional differences might be a result of sampling bias, length-selective fishing mortality, environmental conditions, or genetic differences. However, tagging evidence from previous studies and the presence of two bio-geographic provinces in the region support the hypothesis of two separate breeding populations. The essential reproductive parameters for H. portusjacksoni population analysis in far-eastern Victoria were determined, using a novel method to estimate fecundity and the ovarian cycle of an egg-laying species. In this region, females have between 6 and 20 pre-ovulatory oocytes (average = 14, n = 29, standard deviation = 3.71) before the reproductive season. The species has an annual reproductive cycle correlated with water temperature, with ovulation starting during late winter to early spring and a ~6 month egg-laying period. The period from the onset of vitellogenesis to ovulation of oocytes is ~18 months.