Cryptosporidium is an important protozoan parasite affecting HIV/AIDS patients. To determine the prevalence and the species of Cryptosporidium spp by developing a rapid and efficient real-time PCR-RFLP test. A cross-sectional study was conducted among HIV-infected adults from Kinshasa, the Democratic Republic of Congo. Stool specimens were examined by microscopic evaluation and real-time PCR-RFLP. Out of 242 HIV-infected adults, 10 (4.1%) cases of Cryptosporidium were identified by microscopic examination. Using PCR-RFLP, the prevalence of Cryptosporidium spp was 5.4% (n = 13). All the 13 cases of Cryptosporidium spp had the stage of AIDS of HIV infection. Extracted DNA was amplified by nested PCR targeting a 1030-bp fragment of the 18s RNA gene. RFLP analysis identified one C. parvum, four C. hominis and one non determined Cryptosporidium. The capacity to detect C. parvum, C. hominis and non-determined Cryptosporidium was present among our HIV-infected patients.