This research aims to isolate and identify autochtonous nitrifying bacteria from tilapia pond in Sleman Yogyakarta Indonesia for future application in aquaculture practices in the region. Bacteria were isolated using a nitrification medium. Bacterial characterization was carried out by non-pathogenic test to tilapia (Oreochromis sp.), and nitrification activity test in a single bacterial fermentation medium for 9 days. Bacterial identification was carried out based on the colony and cell morphologies, biochemical tests, and molecular analysis using the 16S rRNA and gyrB genes. A total of 15 isolates of nitrifying bacteria were obtained. Four non-pathogenic isolates obtained the highest nitrification activity on the sixth day of incubation, with nitrate production of 17.26-21.54 ppm. Two selected bacteria, isolates A2 and A3, have colony morphology that is milky white, smooth surface, circular shape, entire edge, and convex elevation. Both bacteria are short rods, Gram-negative, non-motile, produce catalase, fermenting glucose, sucrose, and lactose, and do not produce oxidase, ornithine decarboxylase, indole, and H2S. Molecular analysis showed that the two isolates had the highest similarity (99.28% and 99.34%) to Klebsiella spp.