Cowpea witchweed [Striga gesnerioides (Willd.) Vatke] is a primary constraint of cowpea [Vigna unguiculata (L.) Walp.] production in West Africa. Previously, seven S. gesnerioides races were classified based upon host specificity and genotypic profiling. Because race number and distribution are dynamic systems influenced by gene flow, genetic drift, and natural selection, a thorough investigation of S. gesnerioides diversity and the effectiveness of known sources of resistance in cowpea is needed to develop varieties with durable and broad-spectrum Striga resistance. In this study, we screened seven cowpea lines against 58 unique S. gesnerioides populations collected from across nine West African countries. Individuals from 10 S. gesnerioides populations were genotyped with simple sequence repeat (SSR) markers. We identified six races of S. gesnerioides based on their parasitism of the seven cowpea lines with known differential resistance genotypes. No cowpea line was resistant to all 58 Striga populations and none of the Striga populations were able to overcome the resistance of all seven lines. A novel race, SG6, of the parasite collected from Kudu, Nigeria, was found to overcome more cowpea resistance genes than any previously reported race. SSR analysis indicates that Striga populations are highly differentiated and genetic relatedness generally corresponds with geographic proximity rather than their host compatibility. Due to the dearth of broad-spectrum resistance found among Striga-resistant cowpea lines, there exists a need to stack multiple Striga resistance genes in order to confer broad-spectrum and durable resistance.