Tobacco has been reported to be infected by Rhizoctonia solani isolates belonging to anastomosis groups 1 through to 5. Ten pathogenic isolates of the fungus were collected from tobacco fields in Italy and France that anastomosed in high frequencies with AG BI tester isolates and in low frequencies with tester isolates of all described subgroups of AG2, although morphology and thiamine requirement of the isolates were similar to AG 2‐1. Biomolecular evaluations by means of electrophoresis of polygalacturonase isozymes and RFLPs of ribosomal DNA internal transcribed spacers were carried out. The isolates shared a common pectic zymogram, distinct from those of AG BI and AG 2‐subgroups, while RFLPs of rDNA‐ITS evidenced a limited genetic variation within the homogeneous group and a closer similarity to AG 2‐1. As far as priority is due to the anastomosis behaviour, the isolates should be ascribed to AG BI. However, tobacco isolates differ from tester strains of the known AG BI in their morphology, thiamine requirement, pathogenicity and biomolecular features. In addition they do not anastomose with both AG 3 and AG 6. Therefore they may represent a new subgroup.