Breeding of banana is hampered by its genetic complexity, structural chromosome rearrangements and different ploidy levels. Various scientific disciplines, including cytogenetics, linkage mapping, and bioinformatics, are helpful tools in characterising cultivars and wild relatives used in crossing programs. Chromosome analysis still plays a pivotal role in studying hybrid sterility and structural and numerical variants. In this study, we describe the optimisation of the chromosome spreading protocol of pollen mother cells focusing on the effects of standard fixation methods, duration of the pectolytic enzyme treatment and advantages of fluorescence microscopy of DAPI stained cell spreads. We demonstrate the benefits of this protocol on meiotic features of five wild diploid Musa acuminata bananas and a diploid (AA) cultivar banana “Rejang”, with particular attention on pairing configurations and chromosome transmission that may be indicative for translocations and inversions. Pollen slides demonstrate regular-shaped spores except “Rejang”, which shows fertile pollen grains of different size and sterile pollen grains, suggesting partial sterility and unreduced gamete formation that likely resulted from restitutional meiotic divisions.