Objectives. To quantify the relationship between the segregation of Black, Indigenous, and Latinx communities and COVID-19 testing sites in populous US cities. Methods. We mapped testing sites as of June 2020 in New York City; Chicago, Illinois; Los Angeles, California; and Houston, Texas; we applied Bayesian methods to estimate the association between testing site location and the proportion of the population that is Black, Latinx, or Indigenous per block group, the smallest unit for which the US Census collects sociodemographic data. Results. In New York City, Chicago, and Houston, the expected number of testing sites decreased by 1.29%, 3.05%, and 1.06%, respectively, for each percentage point increase in the Black population. In Chicago, Houston, and Los Angeles, testing sites decreased by 5.64%, 1.95%, and 1.69%, respectively, for each percentage point increase in the Latinx population. Conclusions. In the largest highly segregated US cities, neighborhoods with more Black and Latinx residents had fewer COVID-19 testing sites, likely limiting these communities' participation in the early response to COVID-19. Public Health Implications. In light of conversations on the ethics of racial vaccine prioritization, authorities should consider structural barriers to COVID-19 control efforts. (Am J Public Health. 2022;112(3):518-526. https://doi.org/10.2105/AJPH.2021.306558).