Fluorochrome staining with chromomycin A 3 (CMA) was used to characterize and compare the CMA banding patterns of chromosomes of Clymenia, Eremocitrus, and Microcitrus, which belong to true citrus fruit trees (Citrinae, Rutaceae). All species had 2n = 18 chromosomes. These chromosomes were classified into six types based on the number and position of CMA-positive bands; A: two telomeric and one proximal band, B: one telomeric and one proximal band, C: two telomeric bands, D: one telomeric band, E: without bands, and Dst: type D with a satellite chromosome. Each species possessed three or four types of chromosomes and unique CMA banding patterns. The CMA banding patterns were 2C + 8D + 8E in Cl. polyandra, 2C + 9D + 7E in E. glauca, 1C + 11D + 6E in M. australis, 1B + 2C + 10D + 5E in M. australasica, 8C + 7D + 2E + 1Dst in M. inodora, 2A + 14D + 2Dst in M. warburgiana, and 2C + 9D + 7E in Sydney hybrid. Chromosome configurations of Cl. polyandra, E. glauca, M. australis, and Sydney hybrid resembled each other. This may indicate a common ground of chromosome configuration in the true citrus fruit trees. On the other hand, variability in Microcitrus chromosomes was also demonstrated.