: We evaluated detection of HIV-1 RNA from dried blood spots (DBS) and oral fluid specimens. Between February 2010 and August 2014, HIV-1 was newly diagnosed in eight (2.6%) study participants who had median blood HIV-1 RNA of 61 500 copies/ml (interquartile range 7500-146 000). RNA was detected in seven (87.5%) DBS and three (37.5%) oral fluid swabs but was not detected in either specimen from one participant. DBS may be a reasonable specimen collection method to detect acute infection.