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Grain hardness is an important quality trait of cereals. In the present work, the particle size index (PSI) was used to assess the variability in grain hardness of thirteen triticale genotypes, three wheat genotypes and one rye genotype grown in two subsequent seasons. The PSI values of the triticale samples ranged between 7.7 to 19.2%, representing a medium hard to a very hard grain. Moreover, the sequencing of genes in the Hardness (Ha) locus revealed a limited genetic variability in the grain hardness genes, namely secaloindoline-a (Sina) and secaloindoline-b (Sinb). Two possible new allelic variants were identified, one for each of the secaloindoline genes. The existing variability in the grain hardness as well as polymorphisms in its candidate genes are a good starting point for efforts in breeding new varieties of triticale with improved grain hardness.