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Ramularia leaf spot caused by the fungus <i>Ramularia collo-cygni</i>, has recently become widespread in Estonian barley fields. Currently, disease control in barley fields relies on SDHI and DMI fungicides, which might be threatened by <i>R. collo-cygni</i> isolates that are well-adapted to fungicide pressure. In a two-year study, 353 <i>R. collo-cygni</i> isolates were collected from spring barley fields in Estonia. A total of 153 <i>R. collo-cygni</i> isolates were examined for sensitivity to azoles (DMIs; prothioconazole-desthio, epoxiconazole, mefentrifluconazole) and succinate dehydrogenase inhibitors (SDHIs; boscalid, fluxapyroxad). Epoxiconazole was the least effective and a new fungicide mefentrifluconazole was the most effective DMI. Among SDHIs, fluxapyroxad was more effective than boscalid. Also, single <i>R. collo-cygni</i> isolates with high resistance to tested fungicides occurred, which could affect fungicide control of the pathogen. The entire collection of <i>R. collo-cygni</i> was analysed for mutations in fungicide target proteins. Six mutations were identified in <i>CYP51</i> gene, the most dominant being I381T, I384T, and S459C. Also, numerous point mutations in the <i>SdhC</i> gene were present. The mutation G143A in strobilurin target protein CytB dominates in over 80% of the <i>R. collo-cygni</i> population, confirming the low efficacy of strobilurin fungicides in barley disease control.