Objectives We aimed to (1) evaluate how frequently the uterus and ovaries of healthy, non-pregnant queens are visible; (2) describe their appearance; (3) take their measurements; and (4) determine intra- and inter-observer variabilities in their measurements. We hypothesised that, using a high-frequency linear probe, the uterus and ovaries could be ultrasonographically visualised during any period of the sexual cycle and with any level of operator expertise. Methods Eight queens were enrolled in the study and the ultrasonographical appearance of their uterus and ovaries assessed with a high-frequency linear probe of 15-19 MHz. The diameter of the uterine horns, body and cervix in transverse and longitudinal sections, and the length of the ovaries were recorded. Three observers of different expertise level participated in the study, and the differences between the separate measurements made per queen were evaluated. Results The ovaries and the entire uterus were visualised in every queen. The ovaries were ovoid structures with submillimetric follicles during anoestrus and additional larger follicles depending on the stage of the cycle. An ovarian pattern suggesting cortex and medulla was observed in half the cases. In the uterus, the serosa was a thin hyperechoic outer rim, and layering was observed in half the cases. The cervix was difficult to identify. The intra- and inter-observer variabilities in the uterine horns and the ovaries were minimal (coefficient of variation [CV] 1.4-4.1%) compared with the differences within the queens (CV 10.9-43.4%). The longitudinal and transverse measurements of the horns and the uterine body were the same. Conclusions and relevance The ovaries and uterine horns in queens are accessible ultrasonographically at any stage of their cycle, and can be measured with low intra- and inter-observer variabilities