Objectives To determine the importance of serum prolactin (PRL) in the detection of pituitary stalk interruption syndrome (PSIS) in children with multiple pituitary hormone deficiency (MPHD). We hypothesized that PRL elevation might be a diagnostic indicator of pituitary stalk pathologies. Methods Clinical, radiological, and laboratory features of the 50 cases of MPHD were studied. Results The median age at presentation of the 50 cases (52%, n=26 were female) was 6.61 (0.02-18.9) years. PSIS was detected in 60% (n=30), pituitary hypoplasia in 32% (n=16), partial empty sella in 6% (n=3), and only 2% (n=1) was reported as normal. Out of 50 patients, 21.3% (n=10) were hypoprolactinemic, 44.7% (n=19) were normoprolactinemic, and 34% (n=16) were hyperprolactinemic. The median PRL value was 27.85 (4.21-130) ng/mL in patients with PSIS and 5.57 (0-41.8) ng/mL in patients without PSIS. Additional hormone deficiencies, especially ACTH and LH were detected in follow-up. Conclusions Patients with normal or high prolactin levels deserve special attention regarding the possibility of PSIS. Furthermore, we emphasize the importance of regular follow-up and monitoring for multiple pituitary hormone deficiencies in all patients with a single pituitary hormone deficiency.