Adjustment of crop phenology to resources and constraints of the production environment is crucial for barley adaptation to dry-land environments. The aims of this study were to characterize (under field and under controlled conditions) the diverse heading responses of barley cultivars grown in Spain, and to identify phenotypic trait-molecular marker associations with restriction fragment length polymorphism (RFLP) probes which are linked to major loci controlling vernalization (Sh and Sh2) and photoperiod (Ppd-H1 and Ppd-H2) sensitivities in barley. Thirty-two cultivars were sown in field trials for 3 years in four locations in northern Spain (10 autumn and nine winter sowings), and for 1 year in southern Spain (autumn sowing), and 2 years in Scotland (spring sowings); they were also studied under four glasshouse treatments, combining presence/absence of vernalization and short/long photoperiod. These cultivars were examined for the presence of RFLP polymorphisms with four probes and three restriction enzymes. Analyses of variance detected a number of associations which were quite consistent with expectations, suggesting that marker-loci associations found in mapping populations are, to some extent, maintained in a germ plasm pool, and may be useful for germ plasm characterization and marker-assisted selection in breeding programmes. Two possible epistatic interactions among the probes were detected, one of them possibly related to a short-day vernalization response.