In October 2010 the occurrence of the stem canker symptoms in rapeseed was observed at the locality Rimski Šančevi in Serbia. Several strains of fungi were isolated from the stem. Morphological characteristics of the isolates were studied on a PDA medium at 25±1oC: growth rate, colour, shape and appearance of colonies and the edge of the colony, the pigment, size, colour and shape of pycnidiospores and pycnidia in 10 isolates (isolated in Serbia K-111, IK-112, K-113, K-114, K-115, K-116, IK-117, K-118, K-119, and K-120). All tested strains had fast growth, the regular form of colonies and poor sporulation on nutrient medium. Pycnidiospores are unicellular, hyaline, and mostly straight, with or without a drop of oil. Molecular identification was performed by the application the PCR technique using primers PN3/PN10. In addition to these 10 isolates, two reference strains obtained from the Centre for Agricultural Studies, Rothamsted, UK, which are marked with L. m (Leptosphaena maculans), L. b (Leptosphaeria biglobosa) and 7 reference isolates originating from Serbia, which are marked K-7, St-16, GS-25, L-5, C-3, LJ-2, S-1 were used. On the basis of DNA amplification with primers PN3 and PN10 tape length was about 580 bp for isolates (L.b, IK-111, K-112, K-113, K-114, K-115, K-116, K-117, K-118, K-119, K-120) while that was 560 bp for other group of isolates (L.m, K-7, St-16, GS-25, L-5, C-3, LJ-2, S-1). This study showed that 10 isolates (K-111, K-112, K-113, IK-114, K-115, K-116, K-117, K-118, K-119, K-120) isolated from the stem of rapeseed belong to the species Leptosphaeria biglobosa.