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<i>Septoria</i> leaf blotch (SLB) caused by the fungus <i>Zymoseptoria tritici</i> is the most aggressive disease of winter wheat in various regions of the world. Over the years, SLB has been managed with fungicides differing in mode of action (MOA), including beta-tubulin, cytochrome b-Qo, and 14-alpha demethylase inhibitors. Concurrently, the fungus developed some level of resistance to all of these fungicides. To determine if accumulation of mutations conferring resistance to multiple MOA impairs the fungus in its ability to cause disease, 11 isolates from 5 countries were subjected to 4 cycles of infection on wheat seedlings. Next generation sequencing analysis showed that 5 isolates carried the E198A mutation that confers resistance to beta-tubulin inhibitors as well as the G143A mutation associated with resistance to Qo fungicides. In addition, they also carried multiple mutations in the CYP51 gene decreasing sensitivity to 14-alpha demethylase inhibitors. Four other isolates differed because they had mutations associated with resistance to one or two fungicide classes but not to all three. The remaining 2 isolates were sensitive to all three fungicide classes. The resistant isolates infected wheat and caused the typical disease symptoms produced by the sensitive isolates. Two isolates resistant to all three fungicide classes were less virulent than the sensitive ones but overall the relationship between resistance phenotype and virulence was unclear.