The present paper demonstrates the development of interspecific hybrids between Manihot esculenta Crantz ssp. esculenta (Mee) and M. esculenta Crantz ssp. flabellifolia (Mef) and paternity analysis using microsatellite markers [simple sequence repeat (SSR)]. Three Mef accessions (FLA005, FLA025V and FLA029V) were used for crosses with varieties of Mee: Saracura, Aipim Bravo, COL 1725, Aipim Rosa, Abóbora, Paraná and PER334. The paternity of the interspecific hybrids was investigated using 24 SSRs. The observed heterozygosity (Ho), polymorphic information content (PIC), probability of identity (PI) and paternity exclusion (PE) were evaluated. The rate of breeding success varied from 17 to 92%, and an average of two pollinations were required for each generated hybrid plant. The Ho value ranged from 0·11 to 0·92, and the PIC value ranged from 0·12 to 0·59. The uneven distribution of allele frequencies was accompanied by a high PI average (0·56). However, the combined PE for 21 loci was 0·99, which allows for the determination of the paternity of the hybrids with good discriminatory power. Of the 74 hybrids evaluated, 0·82 had their paternity confirmed using microsatellite markers. Discriminant analysis of principal components (DAPC) indicated the presence of eight clusters, of which, one was composed of only Mef varieties and the supposed hybrid Fla52Sar-H7, which was a product of apomixis. The parent Mee and hybrids were allocated in the other seven clusters. The data obtained demonstrate that SSR markers can be routinely used in breeding programmes to verify the paternity of interspecific crosses of cassava.