This study aimed to examine the relationship between throwing velocity and shoulder range of motion and muscle strength in collegiate male water polo players with and without shoulder pain. Twenty-one players from a Division 2 university league team were divided into a pain group (9 participants) and a non-pain group (12 participants). The relationship between throwing velocity, shoulder range of motion, and muscle strength was assessed. In the non-pain group, the difference in internal rotation strength between the throwing and non-throwing sides significantly correlated with throwing velocity (r = 0.60, p = 0.04). The pain group had a significantly greater external rotation and internal rotation range of motion in the throwing shoulder compared to the non-pain group (mean difference: 15.02 and 15.83, p = 0.02 and 0.04, respectively). These findings suggest that improving internal rotation strength and monitoring shoulder rotation range of motion are crucial for conditioning collegiate water polo players, with consideration for shoulder pain.