SUMMARYTo examine the genetic basis for the variable susceptibility of Striga hermonthica from differing zones of sub‐Saharan Africa to Fusarium oxysporum f. sp. strigae (Fos) isolates Foxy‐2 and FK3, 10 S. hermonthica populations from Eastern and Western Africa were phenotyped for their susceptibility response to Foxy‐2 and FK3, and then genotyped with 22 simple sequence repeat (SSR) markers. There is low genetic differentiation between East African and West African S. hermonthica populations (i.e. the proportion of the total genetic variance contained in the subpopulation relative to the total genetic variance, FST = 0.012, P < 0.05), but intermediate genetic differentiation (FST = 0.143, P < 0.01) underlies the S. hermonthica groups that are differentiated by their phenotypic responses to Fos isolates. An expressed sequence tag SSR (EST‐SSR) marker Y53 (P < 0.01) and a genomic SSR marker E1009 (P < 0.05) were associated with the S. hermonthica class susceptible to Foxy‐2 and FK3 (group A). A divergent S. hermonthica class, consisting of groups with intermediate susceptibility to Foxy‐2 (group B) and susceptibility to either FK3 (group C) or Foxy‐2 (group D), showed no marker–trait association, instead demonstrated linkage disequilibrium decay. Owing to point substitutions and insertion–deletion mutations, the unique, protein‐coding nucleotide sequence at the E1009 locus in group A was partly dissimilar to group B, but was totally distinct from groups C and D. These findings implied that the inconsistent effectiveness of a Fos isolate is better explained by genomic variation in S. hermonthica, rather than by S. hermonthica sampling zones.