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Banana cultivars of agronomic interest have been genetically characterized using two different molecular markers. On the one hand, a panel of 14 trinucleotide single sequence repeats (SSRs or microsatellites) was optimized for homogeneous PCR conditions. It was tested with 50 individuals from seven cultivars, yielding 76 alleles and 5.4 ± 1.8 alleles per locus, while the presence of cultivar‐exclusive alleles allowed the discrimination of all cultivars. On the other hand, a retrotransposon‐based marker system named inter‐primer binding site (iPBS) was implemented for the first time in the Musa genus. A total of 120 bands were detected in eight different Musa cultivars, from which 65.8% were polymorphic and 23.3% were cultivar exclusive. Both techniques allowed a cut‐off identification of all cultivars studied, but overall, iPBS analysis was a more straightforward and economical choice. Despite the fact that we were unable to distinguish local banana varieties belonging to the same cultivar, new cultivar‐specific molecular markers have been developed for the Musa genus, which could be used to guide new breeding programmes and maintain high quality of Plátano de Canarias. Microsatellites (single sequence repeat [SSR] genotyping) and retrotransposon‐based (inter‐primer binding site [iPBS]) markers have been implemented for genetic characterization of banana cultivars with high agronomic interest. Both techniques allowed a cut‐off identification at cultivar level and cultivar‐specific molecular markers have been developed for the Musa genus, which could be used to guide new breeding programmes. [ABSTRACT FROM AUTHOR]