Alternaria tenuissima, a causal agent of violet leaf spots (GVLS) on garlic (Allium sativum L.), causes a massive loss of yields. Boscalid [abbreviated as Bos] has been registered to control the GVLS in China since 2012. Of the 53 isolates separated from Pizhou City of Jiangsu Province in China, 6 isolates exhibited Boscalid-resistant (BosR), the different amino acid substitutions were identified in AtSdh in the BosR isolates (AtSdhBE218G, AtSdhDA164T and AtSdhDV17A+Q18H+A47T). Most BosR isolates exhibited defect on fitness defined as mycelial growth, condition, and virulence. Furthermore, the data revealed that AtSdh expressions, AtSdh enzymatic activity and ATP accumulation were involved in BosR. Positive cross-resistance between Bos and flubeneteram was verified by Spearman rank correlation analysis. Moreover, the resistance mechanism was explored by AlphaFold3 structure prediction that amino acid substitutions at AtSdh were associated to SDHIs-resistance in A. tenuissima. This study revealed novel SDHI-binding sites in Alternaria spp. and highlights the role of amino acid substitutions in SDHIs resistance.