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The Coronavirus Disease 2019 (COVID-19) pandemic has disproportionately impacted people who use drugs (PWUD). This study explored relationships between drug use, COVID-19 testing, vaccination, and infection. This cross-sectional study was conducted in Miami, Florida between March 2021 and October 2022 as part of the National Institutes of Health (NIH) Rapid Acceleration of Diagnostics-Underserved Populations (RADx-UP) initiative and the Miami Adult Studies on HIV (MASH) cohort. Users of cannabis, cocaine/crack, heroin/fentanyl, methamphetamines, hallucinogens, and/or prescription drug misuse in the previous 12 months were considered PWUD. Sociodemographic data, COVID-19 testing history, and vaccination-related beliefs were self-reported. Vaccinations were confirmed with medical records and positivity was determined with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. Statistical analyses included chi-square tests and logistic regression. Of 1,780 participants, median age was 57 years, 50.7% were male, 50.2% Non-Hispanic Black, and 66.0% reported an annual income less than $15,000. Nearly 28.0% used drugs. PWUD were less likely than non-users to self-report ever testing positive for SARS-CoV-2 (14.7% vs. 21.0%, p = 0.006). However, 2.6% of participants tested positive for SARS-CoV-2, with no significant differences between PWUD and non-users (3.7% vs. 2.2%, p = 0.076). PWUD were more likely than non-users to experience difficulties accessing testing (10.2% vs. 7.1%, p = 0.033), vaccine hesitancy (58.9% vs. 43.4%, p = 0.002) and had lower odds of receiving any dose of a COVID-19 vaccine compared to non-users (aOR, 0.63; 95% CI, 0.49–0.81; p<0.001). PWUD presented with greater difficulties accessing COVID-19 testing, greater vaccine hesitancy, and lower odds of vaccination. Testing and immunization plans that are tailored to the needs of PWUD and consider access, trust-building campaigns, and education may be needed. [ABSTRACT FROM AUTHOR]