Although research involving biospecimens is essential in advancing cancer research, minorities, especially African-Americans, are underrepresented in such research. We conducted a mixed-method (qualitative focus groups among African-Americans and quantitative cross-sectional surveys) study on factors associated with biospecimen knowledge and donation intent in the medically underserved urban communities in Southeast and Southwest Washington, DC. Focus groups were conducted among 41 African-Americans and survey data was available from 302 community residents of different races/ethnicities using convenience sampling. We used logistic regression to model the association between biospecimen knowledge and donation intent with selected sociodemographic variables using survey data. Only 47 % of the participants had knowledge of the different types of biospecimens. In multivariate logistic regression models, male gender, African-American race, and low education levels were significantly associated with lower knowledge about biospecimens. Compared to Whites (79 %), fewer AfricanAmericans (39 %) and Hispanics (57 %) had knowledge of biospecimens but the difference was significant for AfricanAmericans only. Positive intent to donate biospecimens for research was observed among 36 % of the survey respondents. After multivariate adjustment, only biospecimen knowledge was associated with donation intent (odds ratio = 1.91, 95 % confidence interval 1.12, 3.27). Contrary to popular opinion, "mistrust of the medical community" was not the most commonly reported barrier for biospecimen donation among African-Americans. "Not knowing how biospecimens will be used" and "lack of knowledge of biospecimens" were the most common barriers. Our study highlights the importance of education on biospecimens among community residents to increase minority participation in biospecimen research.