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The study of the variations in Macrophomina phaseolina, the causal agent of charcoal rot, is critical in understanding the ecology of the isolates and the epidemiology of the diseases they cause. This study sought to identify and characterize M. phaseolina isolates using both molecular and morphological approaches and to determine the virulence of the isolates. Macrophomina phaseolina was isolated from cowpea-diseased stem tissues. Eleven M. phaseolina isolates from the major cowpea growing areas (Manga, Nyankpala, Yendi, Damongo, Tumu, Wa, Akomadan and Ejura) were morphologically and molecularly characterized. DNA of the M. phaseolina isolates were amplified using species-specific PCR primers MpKF1 (5'-CCGCCAGAGGACTATCAAAC-3') and MpKR1 (5'-CGTCCGAAGCGAGGTGTATT-3') and sequencing of the M. phaseolina specific amplicons was obtained. This was followed by searching against the NCBI nucleotide sequence database. Isolates were tested for their pathogenicity on a susceptible cowpea cultivar (IT97K-499-35). Virulence of the M. phaseolina isolates was expressed as the percentage of sown seeds that expressed seed rot and damping-off symptoms 14 days after planting. All isolates were confirmed as M. phaseolina based on the amplicon size obtained, and the sequenced amplicons showed an average of 99% homology to the available M. phaseolina sequences. The radial growth of isolates and the percentage of seed rot and damping off they caused on the IT97K-499-35 cultivar varied significantly. The percentage seed rot and damping off were positively correlated (r = 0.74 and 0.75) with the radial growth and colour intensity of the isolates, respectively. Macrophomina phaseolina infection and disease expression may be predicted by the colour and colour intensity of isolates as well as cowpea genotypes. Isolate Mp_3Mc (dense black colour) was the most virulent while isolate Mp_6Ec (grey colour) was the least virulent, resulting in 76.7% and 26.7% seed rot and damping off, respectively. The three clades resolved through the phylogenetic tree matched the distinct colour types of M. phaseolina which correlated with virulence. Isolates of M. phaseolina are diverse and differ in their pathogenicity. Efforts must be geared towards developing cowpea varieties that have resistance to charcoal rot disease. [ABSTRACT FROM AUTHOR]