Background The 20-m shuttle run test (MSRT) is a common field test used to measure aerobic fitness in controlled environments. The U.S. Army currently assesses aerobic fitness with the two-mile run (TMR), but external factors may impact test performance. The aim of this study is to examine the relationship between the Army Physical Fitness Test TMR performance and the MSRT in military personnel. Methods A group of 531 (403 males and 128 females) active duty soldiers (age: 24.0 ± 4.1 years) performed the MSRT in an indoor facility. Heart rate was monitored for the duration of the test. Post-heart rate and age-predicted maximal heart rate were utilized to determine near-maximal performance on the MSRT. The soldiers provided their most recent Army Physical Fitness Test TMR time (min). A Pearson correlation and multiple linear regression analyses were performed to examine the relationship between TMR time (min) and MSRT score (total number of shuttles completed). The study was approved by the Human Use Review Committee at the U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts. Findings A significant, negative correlation exists between TMR time and MSRT score (r = -0.75, p < 0.001). Sex and MSRT score significantly predicted TMR time (adjusted R2 = 0.65, standard error of estimate = 0.97, p < 0.001) with a 95% ratio limits of agreement of ±12.6%. The resulting equation is: TMR = 17.736-2.464 × (sex) - 0.050 × (MSRT) - 0.026 × (MSRT × sex) for predicted TMR time. Males equal zero, females equal one, and MSRT score is the total number of shuttles completed. Discussion The MSRT is a strong predictor of the TMR and should be considered as a diagnostic tool when assessing aerobic fitness in active duty soldiers.