Majalaya Carp (Cyprinus carpio L.) is one of the most important aquaculture species in Indonesia. This freshwater fish has a great opportunity to be developed, thus, the cultivation data, starting from data on hatchery, nursery, enlargement, to harvest, are available. However, cytogenetic studies of Majalaya carp has been limited. One of the challenges for the chromosomal study is preparing a well-spread chromosome. The objective of this research was to optimize the preparation method to study the chromosome of Majalaya carp from gills. The optimized steps included synchronization (1, 2, 3 h of colcemid treatment); hydrolysis (8, 10, 15 min of KCl treatment); and fixation (1, 2, 3 times). The results showed that the optimum colcemid treatment to synchronize cells at metaphase, KCl incubation for hydrolysis, fixation, and staining method were 2 h, 8 min, and once fixative respectively. By using these optimum conditions obtained from this study, chromosomes of Majalaya carp could successfully be obtained. The results of this study could give insight into further chromosomal analysis.