Learning and memory deficits have been described in rats and mice after ovariectomy (OVX) and across the estrous cycle. Preclinical researchers therefore often avoid using female animals and, consequently, a large male bias exists in the preclinical cognitive literature. In the present study we examined the role of sex hormones in the touchscreen operant platform using the spatial working memory trial unique nonmatching-to-location (TUNL) task. Twenty-nine Long Evans rats were trained to acquire the TUNL task including three incremental spatial separations (S0, S1, S2). Following 20 consecutive days of training, subjects in experiment 1 (n=15) remained intact and immediately progressed to TUNL testing, while subjects in experiment 2 were OVX (n=6) or sham-operated (n=8) prior to testing. Subjects were tested on 4 spatial separations (S0-3) with a 1s or 6s delay between the sample and nonmatching stimuli. The estrous cycle of intact rats was monitored during the 4weeks of testing. The estrous cycle phase did not significantly affect performance. In contrast, compared to intact rats, OVX impaired performance at larger spatial separations (S2-3) during the 1s delay condition. Further, during the 6s delay, OVX impaired S2 performance, however not S3. Our results suggest a probable shift in cognitive strategy following OVX, when tested with a large and novel spatial separation. Our findings suggest that ovarian hormone deprivation following OVX, but not estrous cycle, impairs spatial working memory as measured by the TUNL task. This research is relevant for future studies utilising the touchscreen TUNL task and for cognitive testing of female rats.