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Knowledge of HIV transmission is a prerequisite to practicing safer behaviors to prevent HIV infections and may be expected to vary by region because of cultural and socioeconomic determinants. A cross-sectional study was conducted in rural Kenya using a standardized questionnaire assessing HIV transmission knowledge, socio-demographic and other characteristics. Participants were recruited from the voluntary counseling and testing clinic and the general hospital population of Moi District Hospital. "High" HIV transmission knowledge scorers (≥ 81%) (Mean score) were compared with "low" scorers (<81%). Bivariate and multivariate logistic regression analyses were performed to examine factors associated with HIV transmission knowledge. Of 214 participants, 70 (33%) were HIV-positive, 104 (49%) were HIV-negative, and 40 (19%) did not know. Factors associated with low knowledge in multivariate analyses were lower education (OR 2.36, CI 1.03-5.46), lower household money on healthcare (OR 2.03, CI 1.28-3.21), higher clinic transportation costs (OR 3.14, CI 1.20-9.82), sex without a condom (OR 2.18, CI 1.12-4.26), positive HIV status vs. negative (OR 2.50, CI 1.22-5.26) and positive HIV status vs. unknown (OR 3.57, CI 1.33-9.09). Mean HIV transmission knowledge score was relatively high; however, a large proportion of patients demonstrated low knowledge. Identifying individuals at risk for low knowledge will support targeted HIV education and prevention programs.