In the Czech Republic, increases in the area sown with oilseed rape (Brassica napus L.) and shifts to intensive crop farming systems have contributed to increased incidence and harmfulness of phoma stem canker. This study comprised a large-scale survey of the occurrence of two closely related causal agents of the disease, Leptosphaeria maculans and L. biglobosa, in oilseed rape tissues and the country-wide distribution of each fungal species. In the 2007–11 growing seasons, 1132 leaves with phoma leaf spot symptoms were sampled; from those, 977 L. maculans-type and 477 L. biglobosa-type leaf spots were sampled and analysed by species-specific PCR without pathogen isolation. There were 1159 leaf spots confirmed as infected by Leptosphaeria spp., with 65% of 907 L. maculans-type leaf spots infected by L. maculans only and 35% co-infected by both species; and with 88% of 252 L. biglobosa-type leaf spots infected by L. biglobosa only and 12% co-infected by both species. Furthermore, 217 monopycnidial isolates were collected from selected leaf spots and identified based on pigment production during solid- and liquid-media culture and PCR assay. Most (82%) isolates originating from L. maculans-type leaf lesions were L. maculans, and most (69%) isolates collected from L. biglobosa-type leaf lesions were L. biglobosa. Co-infection by both species was found in both L. maculans-type and L. biglobosa-type leaf lesions. In 2007–12, 708 stems with phoma stem canker symptoms and 2635 plant tissues from upper stem, stem base, root collar and taproot of each stem were sampled for PCR; symptoms on the four parts of each stem were assessed before taking tissue samples. There were 1495 plant tissues confirmed as infected by Leptosphaeria spp., with the proportion of plant tissue in which only L. biglobosa DNA (62%) was amplified greater than that with only L. maculans DNA (11%) or with both L. maculans and L. biglobosa DNA (27%). Although both species were detected in leaf samples in autumn, L. biglobosa was the more frequently detected species in stem samples in summer, suggesting that L. biglobosa is the more successful in colonising oilseed rape tissues in later growth stages of the plant in the Czech Republic.