Chromosome preparation from fish embryos is a quick, cheap and simple method. However, it involves a fixation step in acetic acid, which has been reported to impair the suitability of chromosomes for certain banding techniques, and some available protocols require the mixing of several embryos to obtain preparations with a sufficient amount of metaphases, which can mask potential interindividual variation. Therefore, the method is used only rarely. Here, we describe a method for the preparation of a high number of high-quality metaphase chromosomes from single acetic acid fixed embryos of Chromaphyosemion killifishes and demonstrate that such preparations can be used routinely for conventional Giemsa staining, C-banding and sequential chromosome banding with chromomycin A3, Giemsa and AgNO3 or with DAPI, chromomycin A3, Giemsa and C-banding, respectively. The protocol is particularly suitable for crossing experiments that require screening of various chromosome markers.