Cowpea [Vigna unguiculata (L.) Walp.] is an important socioeconomic crop in Brazil, mainly in the Northeast and more recently in the Midwest of Brazil. Charcoal rot caused by Macrophomina phaseolina (Tassi) Goid, is an important disease in semiarid regions, where edaphoclimatic conditions are favorable to the development of disease. The aim of this study was to evaluate the response of 100 cowpea lines to two isolates of M. phaseolina. The experiments were conducted in a completely randomized design, with five replications (two plants per pot). The main variables evaluated were lesion length and relative growth compared to control (RGCC). Among the evaluated accessions, 15% of the lines were resistant to isolate 59 and 11% of the lines were resistant to isolate CMM 2106 of M. phaseolina. Therefore, these accessions can be used as a source of resistance to M. phaseolina by farmers directly as new cultivars or in future hybridizations of cowpea genetic breeding programs.