Phytoplasmosis associated with the ‘Candidatus Phytoplasma solani’ subgroups 16SrXII-A and 16SrXII-P (Mollicutes) poses a threat to European sugar beet cultivation, alongside the “basses richesses” syndrome caused by ‘Ca. Arsenophonus phytopathogenicus’ (Gammaproteobacteria). Both subgroups co-occur in sugar beet and have been detected in Pentastiridius leporinus, a polyphagous planthopper associated with SBR-affected fields. Despite ongoing monitoring of disease spread and pathogen characterization, approaches for direct subgroup differentiation remain limited. Consequently, discriminative conventional PCR assays targeting secY were developed, with detection limits estimated using synthetic gene fragments. Assay performance was assessed using 900 DNA templates from sugar beet from Germany and neighboring countries, 50 German red beet samples, and 320 planthoppers. Distinct amplicon sizes of 549 bp and 857 bp enabled reliable detection and differentiation of 16SrXII-A and 16SrXII-P, respectively. Detection rates were broadly concordant with those obtained using an established phytoplasma detection approach. Estimated detection limits were 100 target copies for 16SrXII-A and 1000 for 16SrXII-P. Assay application indicated more frequent detection of 16SrXII-P in samples from Germany and of 16SrXII-A in those from neighboring countries. The assays provide a rapid and accessible tool for subgroup differentiation and may facilitate large-scale epidemiological surveillance and future studies of host tolerance.