Uncultivable bacteria from the genus ‘Candidatus Phytoplasma’ are associated with grapevine yellows (GY) diseases worldwide. In Euro-Mediterranean viticultural areas, GY are most frequently caused by Bois Noir (BN) and Flavescence Dorée (FD) phytoplasmas. Surveys of GY in Croatia have been conducted regularly since 1997. BN phytoplasmas have been found to be widespread, while FD phytoplasmas were recently discovered in restricted areas of the country. The aim of this study was to assess the variability of genotypes involved in GY pathosystems by a multilocus sequence typing (MLST) approach. Grapevine, weed and insect vector samples were collected from three locations. Species-specific stamp and vmp1 genes, together with house-keeping genes tuf and secY, were amplified from BN strains. In FD strains, the genes secY, map and uvrB-degV were analyzed. MLST revealed a diversity of BN genotypes, one of which was prevalent and identified in samples of grapevine and the insect vector Hyalesthes obsoletus, corroborating their affiliation to the same pathosystem. Distinct BN strains found in bindweed and two grapevine samples indicated the presence of different BN pathosystems involving a yet unidentified vector, possibly from the genus Reptalus. Moreover, a co-occurence of BN and FD phytoplasma in the same vineyard was identified. The genotyping of FD strains from both grapevine and Scaphoideus titanus showed the presence of at least two distinct FD genotypes at two different locations, suggesting separate introductions of the disease into the country. In this study, MLST proved to be a useful and informative tool in advancing the understanding of GY epidemiology in Croatia.