Abstract'Candidatus Phytoplasma solani’ (CPs), a phytoplasma endemic to the Euro-Mediterranean basin is a causative agent of several plant diseases, including the grapevine yellows disease “bois noir” (BN). As different CPs strains have been shown to have different ecological reservoirs and pathways for spread, the genetic characterization of CPs strains is a prerequisite, and better control of BN relies on the identification of reservoir plants. The variability of the phytoplasma genotypes involved in the BN pathosystem in Croatian vineyards was assessed by a multilocus sequence typing (MLST) approach. The genotyping was performed on selected grapevine, wild plants, and insects collected within the eleven years of national survey conducted in all Croatian viticultural regions. The extensive tuf, secY, stamp, and vmp1 genes-based MLST analyses revealed two new genotypes for stamp and vmp1 genes, designated as ST59 and V28, respectively, and overall identified 28 different CPs MLST genotypes. The prevalent MLST genotype in grapevine CPsSqt21 (S6/ST6/V18/tuf-b2) was widespread in nine counties across Uplands, Slavonia, and Danube wine regions and was affiliated to the known vector Hyalesthes obsoletus and to Urtica dioica. The other two most frequent genotypes were the U. dioica-associated CPsSqt28 (S39/ST46/V3/tuf-a) and the C. arvensis-associated CPsSqt2 (S1/ST9/V4/tuf-b1). CPs of different vmp1 genotypes was also detected in Cixius wagneri specimens originating from different parts of Croatia. In addition, CPs was detected in several Dichtyophara europaea insects and in two new potential plant reservoirs Ailanthus altissima and Robinia pseudoacacia. The substantial number of found MLST genotypes indicates the presence of several independent epidemiological cycles and is certainly a consequence of a unique geographical position of Croatia, bridging the different eco-climatic areas of central and south-eastern Europe.