Barriers to gene flow that act after mating but before fertilization are often overlooked in studies of reproductive isolation. Where species are sympatric, such “cryptic” isolating barriers may be important in maintaining species as distinct entities. Drosophila yakuba and its sister species D. santomea have overlapping ranges on the island of São Tomé, off the coast of West Africa. Previous studies have shown that the two species are strongly sexually isolated. However, the degree of sexual isolation observed in the laboratory cannot explain the low frequency (∼1%) of hybrids observed in nature. This study identifies two “cryptic” isolating barriers that may further reduce gene flow between D. yakuba and D. santomea where they are sympatric. First, noncompetitive gametic isolation has evolved between D. yakuba and D. santomea: heterospecific matings between the two species produce significantly fewer offspring than do conspecific matings. Second, conspecific sperm precedence (CSP) occurs when D. yakuba females mate with conspecific and heterospecific males. However, CSP is asymmetrical: D. santomea females do not show patterns of sperm usage consistent with CSP. Drosophila yakuba and D. santomea females also differ with respect to remating propensity after first mating with conspecific males. These results suggest that noncompetitive and competitive gametic isolating barriers may contribute to reproductive isolation between D. yakuba and D. santomea.