Syringe-sharing behaviors among injection drug users (IDUs) are typically based on self-reports and subject to socially desirable responding. We used 3 short tandem repeat (STR) genetic biomarkers to detect sharing in 2512 syringes exchanged by 315 IDUs in the Baltimore needle exchange program (NEP; 738 person-visits). Demographic characteristics as well as direct and indirect needle-sharing behaviors corresponding to the closest AIDS Link to Intravenous Experience (ALIVE) study visits were examined for association with multiperson use (MPU) of syringes. Overall, 56% of the syringes exchanged at the Baltimore NEP had evidence of MPU. Less MPU of syringes (48% vs. 71%; P < 0.0001) was seen with more rapid syringe turnaround (<3 days). IDUs always exchanging their own syringes (“primary” syringes) were less likely to return syringes with evidence of MPU (52%) than those who exchanged syringes for others (“secondary” syringes; 64%; P = 0.0001) and those exchanging primary and secondary syringes (58%; P = 0.004). In a multivariate analysis restricted to primary exchangers, MPU of syringes was associated with sharing cotton (adjusted odds ratio [AOR] = 2.06, 95% confidence interval [CI]: 1.30 to 3.28), lending syringes (AOR = 1.70, 95% CI: 1.24 to 2.34), and injecting less than daily (AOR = 0.64, 95% CI: 0.43 to 0.95). These findings support additional public health interventions such as expanded syringe access to prevent HIV and other blood-borne infections. Testing of STRs represents a promising approach to examining and accessing complex behavioral data, including syringe sharing.