Root lesion nematodes, particularly Pratylenchus goodeyi, are among the major pests affecting banana production in Tanzania. In this study, morphological, morphometric and molecular approaches were employed to identify nematodes from banana-growing regions. Eighty P. goodeyi isolates were identified and confirmed through analysis of three DNA regions. The P. goodeyi isolates in this study showed kinship with P. goodeyi from other parts of the world and other sequences in the GenBank database. The results from this study will provide an insight into the presence of harmful root lesion nematodes and can inform nematode management strategies in Tanzania.