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In contrast to the long-lasting taxonomic classification of <i>Plenodomus lingam</i> and <i>P. biglobosus</i> as one species, formerly termed <i>Leptosphaeria maculans</i>, both species form separate monophyletic groups, comprising sub-classes, differing considerably with epidemiology towards Brassicaceae plants. Considering the great differences between <i>P. lingam</i> and <i>P. biglobosus</i>, we hypothesized their metabolic capacities vary to a great extent. The experiment was done using the FF microplates (Biolog Inc., Hayward, CA, USA) containing 95 carbon sources and tetrazolium dye. The fungi <i>P. lingam</i> and <i>P. biglobosus</i> subclade ‘brassicae’ (3 isolates per group) were cultured on PDA medium for 6 weeks at 20 °C and then fungal spores were used as inoculum of microplates. The test was carried out in triplicate. We have demonstrated that substrate richness, calculated as the number of utilized substrates (measured at λ490 nm), and the number of substrates allowing effective growth of the isolates (λ750 nm), showed significant differences among tested species. The most efficient isolate of <i>P. lingam</i> utilized 36 carbon sources, whereas <i>P. biglobosus</i> utilized 60 substrates. Among them, 25–29 carbon sources for <i>P. lingam</i> and 34–48 substrates for <i>P. biglobosus</i> were efficiently used, allowing their growth. Cluster analysis based on Senath criteria divided <i>P. biglobosus</i> into two groups and <i>P. lingam</i> isolates formed one group (33% similarity). We deduce the similarities between the tested species help them coexist on the same host plant and the differences greatly contribute to their different lifestyles, with <i>P. biglobosus</i> being less specialized and <i>P. lingam</i> coevolving more strictly with the host plant.