BackgroundLongitudinal evaluation of plasma endogenous ACTH concentration in clinically normal horses has not been investigated in the Southern Hemisphere.ObjectivesTo longitudinally determine monthly upper reference limits for plasma ACTH in 2 disparate Australian geographic locations and to examine whether location affected the circannual rhythm of endogenous ACTH in the 2 groups of horses over a 12‐month period.AnimalsClinically normal horses <20 years of age from 4 properties (institutional herd and client owned animals) in Perth (n = 40) and Townsville (n = 41) were included in the study.MethodsA prospective longitudinal descriptive study to determine the upper reference limit and confidence intervals for plasma ACTH in each geographic location using the ASVCP reference interval (RI) guidelines, for individual months and monthly groupings for 12 consecutive months.ResultsPlasma endogenous ACTH concentrations demonstrated a circannual rhythm. The increase in endogenous ACTH was not confined to the autumnal months but was associated with changes in photoperiod. During the quiescent period, plasma ACTH concentrations were lower, ≤43 pg/mL (upper limit of the 90% confidence interval (CI)) in horses from Perth and ≤67 pg/mL (upper limit of the 90% CI) in horses from Townsville, than at the acrophase, ≤94 pg/mL (upper limit of the 90% CI) in horses from Perth, ≤101 pg/mL (upper limit of the 90% CI) in horses from Townsville.Conclusions and Clinical ImportanceCircannual rhythms of endogenous ACTH concentrations vary between geographic locations, this could be due to changes in photoperiod or other unknown factors, and upper reference limits should be determined for specific locations.