Festulolium are intergeneric hybrids developed to combine the agronomic advantages of ryegrass (Lolium) with the persistence and abiotic stress tolerance of fescues (Festuca). To survey genetic evolution of tetraploid Festulolium populations bred from F glaucescens (Fg) x L. multiflorum (Lm) cross, we examined 47 EST-SSR and STS public markers for: their amplification efficiency in Fg and Lm; the genome specificity of their PCR products; and ability for high throughput genotyping (band size <= 500 bp). A total of 322 bands were scored among which 93 were Fg-specific, 173, Lm-specific and 56 common to both species. F. arundinacea originating EST-SSR amplified Fg DNA at a significant higher rate than L. perenne EST-SSR and showed a significant higher number of Fg-specific bands. In fine, we selected 12 markers among which two primer pairs need to be redefined for use in ABI sequencer. We also present the genotypic distribution of 7 markers in a population of tetraploid Lm x Fg F1 hybrids.