Abstract Background Melissa officinalis (balm) is a perennial medicinal. Climate change and high cultivation costs necessitate the breeding of new cultivars with improved stress tolerance and high metabolite content. However, the high costs of phenotyping large collections limit breeding progress. This study characterized the genetic diversity and population structure of 256 genotypes from 215 international accessions using flow cytometry and genotyping by sequencing (GBS). The primary objective was to identify untapped genetic resources and establish core sets to streamline future breeding and research efforts. Results Morphological characterization and flow cytometry revealed a clear division by ploidy and subspecies. The collection comprised 209 diploid and three polyploid genotypes of ssp. officinalis (2C = 1.79 pg) and 44 tetraploid genotypes of ssp. altissima (4C = 3.57 pg). GBS generated 29,307 and 9,909 SNPs for the entire collection and a subset of ssp. officinalis genotypes, respectively. We identified significant genetic differentiation between the subspecies, as confirmed by PCA (PC1 = 69.9%), STRUCTURE, and hierarchical clustering. With 21,770 private alleles and HE = 0.38 subspecies altissima exhibited greater genetic divergence than ssp. officinalis (2,953 private alleles, HE = 0.07). Within ssp. officinalis, several clusters of untapped genetic diversity from Armenia, France, Georgia, and Spain were identified. Furthermore, genomic analysis revealed inconsistencies in varietal purity in three out of four investigated traditional cultivars, where genotypes bearing the same name were assigned to different genetic clusters. Based on a clustering approach, two core sets were constructed. The first core set (Core15, 5.7% of the collection) of 9 officinalis and 6 altissima genotypes aimed to comprise the diversity of the entire collection with minimal redundancy for long-term breeding programs. An extended core set of 30 genotypes (Core30, 11.3%) focused on the subspecies officinalis and included more cultivated material. Conclusions This study provides a genomic characterization of a comprehensive M. officinalis collection. The identification of distinct genetic clusters and the development of optimized core sets significantly reduce the resources required for future evaluations. These core sets serve as a robust foundation for breeding programs aimed at enhancing agronomic traits such as drought tolerance, while increasing pharmaceutical quality.
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