Objective: To establish statistically valid, population-based reference intervals (RIs) for canine anti-Müllerian hormone (AMH) and define changes in AMH and inhibin-B in bitches during breeding cycles., Methods: A homologous canine ELISA was used to measure AMH in serum samples (collected between May 2019 and July 2024) from 102 intact and 78 reportedly ovariohysterectomized (OVH) bitches and 8 bitches before and after ovariohysterectomy, and in longitudinal samples from 24 bitches undergoing breeding management. Established 95% RIs were used in a retrospective assessment of 3,193 clinical submissions. Cyclic variation of AMH and inhibin-B (heterologous ELISA) were regressed with time and normalized to the rise in progesterone in samples from breeding bitches., Results: Intact and OVH RIs for AMH were calculated with and without inclusion of 7 samples from reportedly OVH bitches that had AMH concentrations in the intact RI. Anti-Müllerian hormone and inhibin-B were positively correlated, and AMH was 3 times higher in proestrus than in estrus. Retrospectively, of 3,193 samples submitted for clinical AMH testing, 41% to 56% were in or above the intact AMH interval, 37% to 44% were within the OVH interval, and < 10% were inconclusive, depending on how RIs were defined., Conclusions: Statistically valid, population-based RIs establish a sound basis for interpreting results of clinical submissions requesting AMH to assess gonadal status in the bitch., Clinical Relevance: Confirmation of cyclic variation in AMH (and, for the first time, inhibin-B) reaffirms proestrus as the optimum time to draw samples, and ≤ 10% of samples submitted for determination of gonadal status are expected to fall in an inconclusive AMH RI.