Understanding calving rates of wild whale populations is critically important for conservation. Reproduction of humpback whales is difficult to monitor and basic physiological information such as pregnancy rates and calving intervals remain unknown. We hypothesized that pregnant whales have sustained elevations in baleen progesterone that temporally correlate with gestation. To test this hypothesis, baleen progesterone profiles from two adult female North Pacific humpbacks, both with extensive sightings records and documented pregnancies, were compared to those of a nulliparous female and a juvenile male. Baleen specimens were collected at necropsy and subsampled every 2 cm. Progesterone was quantified using enzyme immunoassays with the date of growth of each sampling location confirmed via stable isotope analysis. Progesterone profiles from both pregnant whales showed sustained high progesterone content in areas corresponding to known pregnancies, inferred from calf sightings or post-mortem data. The younger female had higher progesterone during pregnancy than the older female but levels during non-pregnancy were similar. The nulliparous female and the male had low progesterone throughout their baleen plates. Baleen hormone analysis can determine how progesterone changes throughout gestation and has potential for estimation of reproductive history and calving intervals.