Pituitary gland (PG) tumors are common in rats over the age of 2. CT and MRI can be difficult to apply in this species, whereas ultrasound is more feasible and useful. To our knowledge, there are no studies on PG ultrasound in rats. The aim of this prospective, analytical study was to evaluate the reliability of ultrasound in identifying PG, to define the ultrasound dimension of the gland in a group of rats with no evidence of pituitary diseases, and to examine its correlations with age, sex, and weight. After localizing the PG with an MRI study on one rat, the gland was identified in 21 rats by ultrasound by two sonographers using a ventral neck approach and a transversal scan with a linear probe. The gland appears as a hypoechoic oval structure with a thin hyperechoic margin. The rats (15 male and 6 female) ranged from 4 to 18 months in age (median 6 months) and from 270 to 640 g in weight (median 370 g). The median pituitary width was 3.96 mm (interquartile range 25–75%: 6–4.5 mm), and the median height was 1.48 mm (interquartile range 25–75%: 1.3–1.67 mm). There was no statistically significant correlation between PG size and rat weight, gender, or age. We believe that these ultrasound measurements could be useful for the diagnosis of pituitary disease, irrespective of whether neurological symptoms are present. We report a clinical case of a rat with a pituitary mass detected by ultrasound and CT.