Cassava root rot is a highly destructive disease that causes great damage in Brazil. Identification of the causal agents is essential for the management of this disease. Roots of cassava plants (Manihot esculenta Crantz) with symptoms of root rot were collected from a cassava crop in the municipality of Garanhuns, Pernambuco State, Brazil. This study aimed to identify a Diaporthe species isolated from cassava root rot in Brazil and evaluate pathogenicity in cassava plants and roots. At 7 days after inoculation (DAI), tuberous roots showed necrotic lesions, a soft appearance, and pink color in the center and brown to greenish at the edges, while seedlings showed root rot, wilting, yellowing, drying out, and leaf fall at 66 DAI in the soil. All plants in the control group were asymptomatic. After reisolations and confirmation of Koch's postulates, the causal agent was conclusively identified as Diaporthe ueckeri through the multilocus phylogenetic analyses involving the ITS, TEF1-α, TUB2, CAL, and HIS genes. The results showed that the morphological characteristics, such as colonies with abundant aerial mycelium, formation of pycnidia and alpha conidia, were consistent with those described for species of the genus Diaporthe. This is the first report of D. ueckeri causing cassava root rot in Brazil and worldwide.