The annual growth line hypothesis predicts that a single, countable growth line is deposited annually in turtles. Support for this hypothesis is implied whenever growth line counts are used to estimate age in turtles. However, the hypothesis has not been thoroughly tested. We tested the annual growth line hypothesis by calibrating growth line counts against elapsed time in a population of red-eared sliders, Trachemys scripta elegans, in central Oklahoma. Thirty-one percent of all individuals (349/1129) had countable growth lines. The maximum number of growth lines that could be reliably counted was four. In each year of the study, the first turtles were captured early in March, yet the first new growth lines were not documented until late April. We recorded 1569 captures of 106 turtles that were captured in consecutive years and had countable growth lines throughout the study. Within these turtles, the annual growth line hypothesis was supported. Growth line counts agreed with elapsed time for 100 (94%) turtles. Three turtles appeared to add two growth lines in one year, and three turtles did not add a growth line in one year. We conclude that growth line counts can be used to age turtles in this population up to age four. However, we recognize several sources of error in growth line counts, and encourage researchers to conduct intensive sampling with repeated growth line counts from individuals.