Triticale is being considered as a bioindustrial crop in Canada using genetic modification. Because related spring wheat (Triticum aestivum) and durum wheat (T. durum) may exhibit synchronous flowering and grow in proximity, determination of interspecific gene flow when triticale is the pollen donor is necessary to evaluate potential risk. Pollen-mediated gene flow risk assessments generally rely on phenotypic markers to detect hybridization but DNA markers could be powerful and less ambiguous in quantifying rare interspecific gene flow. Six cultivars representing four species [spring wheat, durum wheat, triticale and rye (Secale cereale)] were screened with 235 spring wheat and 27 rye SSR markers to evaluate transferability and polymorphism. Fifty-five polymorphic markers were used in conjunction with morphological characterization to quantify interspecific gene flow from a blue aleurone (BA) triticale line to two spring wheat cultivars (AC Barrie and AC Crystal) and one durum wheat cultivar (AC Avonlea). Approximately 1.9 Million seeds from small plot experiments were visually screened in comparison with known hybrid seed. In total 2031 putative hybrids were identified and 448 germinated. Morphological analysis of putative hybrid plants identified five hybrids while molecular analysis identified 11 hybrids and two were common to both. Combined, 14 hybrids were confirmed: 10 spring wheat × triticale (0.0008 % of harvested seed): seven AC Barrie × BA triticale (0.001 %) and three AC Crystal × BA triticale (0.0005 %); and four durum wheat × triticale (0.0006 %). The occurrence of rare hybrids does not present a substantial risk to the development of GM triticale.