BackgroundFeline sporotrichosis is common in Malaysia. Thermosensitivity and effects of azole treatment on fungal susceptibility are unknown.ObjectivesTo evaluate thermotolerance and antifungal susceptibility of feline Malaysian Sporothrix isolates, compare microdilution (MD) and E‐test results, and investigate changes in susceptibility during azole therapy.MethodsSporothrix schenckii sensu stricto was isolated from 44 cats. Thermotolerance was determined via culture at 37°C for 7 days. Susceptibility to itraconazole (ITZ), ketoconazole (KTZ) and terbinafine (TRB) was assessed in 40 isolates by MD; to amphotericin B (AMB), KTZ, ITZ, fluconazole (FLC) and posaconazole (POS) by E‐test. Results were statistically compared by Pearson's Product Moment. In eight ketoconazole treated cats, susceptibility testing to itraconazole and ketoconazole was repeated every two months for six months.ResultsThermotolerance was observed in 36 of 44 (82%) isolates. Assuming that isolates growing at antifungal concentrations ≥4 mg/mL were resistant, all were resistant on E‐test to FLC and AMB, 11 (28%) to POS, 6 (15%) to ITZ and 1 (3%) to KTZ. On MD, 27 of 40 (68%) were resistant to TRB, 2 (5%) to ITZ and 3 (8%) to KTZ. There was no correlation between E‐test and MD results (KTZ r = 0.10, P = 0.54, and ITZ r = 0.11, P = 0.48). MD values for ITZ and KTZ did not exceed 4 mg/L during KTZ therapy.ConclusionThe majority of feline isolates in Malaysia are thermosensitive. Lack of correlation between E‐test and MD suggests that the E‐test is unreliable to test antifungal susceptibility for Sporothrix spp. compared to MD. KTZ was the antifungal drug with the lowest MIC. Prolonged KTZ administration may not induce changes in antifungal susceptibility.