Abstract The field of marine mammal conservation has dramatically benefited from the rapid advancement of methods to assess the reproductive physiology of individuals and populations from steroid hormones isolated from minimally invasive skin–blubber biopsy samples. Historically, this vital information was only available from complete anatomical and physiological investigations of samples collected during commercial or indigenous whaling. Humpback whales (Megaptera novaeangliae) are a migratory, cosmopolitan species that reproduce in warm, low-latitude breeding grounds. New Caledonia is seasonally visited by a small breeding sub-stock of humpback whales, forming part of the endangered Oceania subpopulation. To better understand the demographic and seasonal patterns of reproductive physiology in humpback whales, we quantified baseline measurements of reproductive hormones (progesterone—P4, testosterone—T and 17β-estradiol—E2) using an extensive archive of skin–blubber biopsy samples collected from female humpback whales in New Caledonia waters between 2016 and 2019 (n = 194). We observed significant differences in the P4, T and E2 concentrations across different demographic groups of female humpback whales, and we described some of the first evidence of the endocrine patterns of estrous in live free-ranging baleen whales. This study is fundamental in its methodological approach to a wild species that has a global distribution, with seasonally distinct life histories. This information will assist in monitoring, managing and conserving this population as global ecological changes continue to occur unhindered.