Purpose The primary objective of this study was to examine and compare lumbar multifidus (LM) muscle size, asymmetry, and function in university football players with and without low back pain (LBP). A secondary objective was to examine the relationship between LM characteristics and body composition in football players. Methods Ultrasound assessments of the LM muscle were performed in 41 university football players during the preseason. LM muscle cross-sectional area, echo intensity (e.g., indicator of fatty infiltration and connective tissue), thickness at rest, and thickness during submaximal contraction (e.g., contralateral arm lift) measurements in prone and standing positions were obtained bilaterally at the L5–S1 level. Body composition measures were acquired using dual-energy x-ray absorptiometry. A self-administered questionnaire was used to obtain LBP history data. Results The LM muscle thickness at rest in prone and in standing was significantly smaller in football players who reported the presence of LBP in the previous 3 months. The LM cross-sectional area in prone was significantly and positively correlated with weight, height, lean body mass, total fat mass, and total percent body fat. LM echo intensity was strongly correlated with total percent body fat and total fat mass and negatively correlated with the percent thickness change during contraction. Conclusion The results of this study provide novel information on LM muscle morphology and activation in football players in prone and standing and suggest that players with LBP in the previous 3 months had smaller LM muscle thickness. LM morphology was strongly correlated with body composition measurements.