Trunk muscles play an important role in providing both mobility and stability during dynamic tasks in athletes. The purpose of this study was to evaluate the within-day and between-day reliability of ultrasound (US) in measuring abdominal and lumbar multifidus muscle (MF) thickness in athletes with and without hamstring strain injury (HSI). Fifteen male soccer players (18-30 years old) with and without HSI were evaluated using two US probes (50 mm linear 7.5 MHZ and 70 mm curvilinear 5 MHz). The abdominal muscle thickness as well as the cross sectional area (CSA) of the MF was measured. To determine within and between days reliabilities, the second and third measurements were repeated with two hours and one week intervals, respectively. Intraclass correlation coefficients for athletes with and without HSI demonstrated good to high reliability for the abdominal muscle thickness (0.82 and 0.93) and CSA of the MF muscle (0.84 and 0.89, respectively). Our results indicated that US seemed to be a reliable instrument to measure abdominal and lumbar multifidus muscle thickness in soccer players with and without HSI. However, further studies are recommended to support the present study findings in other athletes.