Hyperprolactinemia is a common disorder of the hypothalamic-pituitary axis, and a cause of ovarian dysfunction in women. Currently, over half of non-cycling African elephant females in North America also are hyperprolactinemic, suggesting a similar link between these two conditions may exist. The objective of this study was to determine the relationship between acyclicity and prolactin status by comparing mean prolactin concentrations of bi-weekly samples collected over a 1-year period in 2012 with 20 years of historical weekly progestagen data to assess cyclicity. Females were categorized as: 1) non-cycling with an average prolactin concentration of 15 ng/ml or greater (HIGH; n = 17); 2) non-cycling with an average prolactin concentration below 15 ng/ml (LOW; n = 16); and 3) typical temporal patterns of progestagen and prolactin secretion (NORMAL; n = 45), and evaluated based on length of time (in years) they had experienced ovarian inactivity. Results showed that the majority of HIGH prolactin elephants had been acyclic for at least 5 years, and in a number of cases (n = 9) for over 10 years. By contrast, most of the LOW prolactin elephants had experienced acyclicity for less than 5 years. Finally, there was a positive association between duration of acyclicity and mean prolactin concentrations, with an increase in the likelihood of having higher prolactin concentrations the longer an individual was acyclic. This study highlights the importance of longitudinal hormonal datasets to examine temporal changes in biological functioning and better understand the etiology of infertility problems.