Koa (Acacia koa), Hawaii’s second most common endemic tree that is ecologically, economically and culturally important to Hawai’i, is under threat from koa wilt disease, particularly in the low- to mid-elevation forests caused by Fusarium oxysporum f. sp. koae (Fo koae). Fusarium oxysporum is a cosmopolitan vascular wilt pathogen of diverse plant hosts. Morphological identification methods have little utility for pathogenic and non-pathogenic F. oxysporum strains because differences are unreliable. After screening for pathogenicity, genetic analyses of 24 isolates showed a clear and well-supported clade of pathogenic Fo koae isolates. Because highly virulent strains of Fo koae are genetically but not morphologically distinct for non-pathogenic isolates, our objectives were to develop a DNA-based probe to detect pathogenic Fo koae and examine population genetics among isolates from different Hawaiian Islands. Using whole-genome sequencing of one highly virulent isolate and one non-pathogenic isolate of Fo koae, a SNP-based DNA probe was developed to identify pathogenic strains of Fo koae. This probe was verified on 21 characterized isolates (16 pathogenic and 5 non-pathogenic). To confirm probe specificity, greenhouse assays were conducted on newly collected Fo koae isolates from across four Hawaiian Islands. The results will provide guidance to koa restoration efforts and resistance-breeding and screening programs for koa.