Objective To understand clinical features and homology of Acinetobacter pittii (A. pittii) and Acinetobacter nosocomialis (A. nosocomialis) infection in a hospital. Methods A total of 335 non-duplicate clinical isolates of Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ACB) were collected from a hospital between January 2016 and December 2016, species were identified by 16S-23S rRNA gene spacer sequence analysis, clinical data and laboratory detection results of A. pittii, A. nosocomialis and A. baumannii were compared, OXA-51 gene of three kinds of bacteria was detected by polymerase chain reaction(PCR), homology between A. pittii and A. nosocomialis was analyzed by randomly amplified polymorphic DNA (RAPD). Results Among 335 ACB strains, 18, 23, 284, and 10 were A. pittii, A. nosocomialis, A. baumannii, and other Acinetobacter respectively. There were significant differences in admission rate of intensive care unit(ICU), invasive operation, pulmonary infection rate, and mortality during hospitalization period between patients with infection of A. baumannii and A. nosocomialis (all P<0.05). Resistance rates of A. pittii and A. nosocomialis to most antimicrobial agents were lower than those of A. baumannii. No positive amplification of OXA-51 gene was found in A. pittii and A. nosocomialis, PCR detection was positive in OXA-51 gene of 284 strains of A. baumannii. A. pittii and A. nosocomialis were divided into four different clones by RAPD detection, homology analysis showed that clone A and clone F were epidemic strains of A. pittii and A. nosocomialis respectively. Conclusion A. pittii and A. nosocomialis should be considered as different clinical strains from A. baumannii, amplification of OXA-51 gene can be used as a simple and rapid molecular biological technique for rapid identification of A. pittii and A. nosocomialis. Surveillance of A. pittii and A. nosocomialis should be strengthened.