Abstract Background Malignant transformation of normal gastric cells is a complex and multistep process, resulting in development of heterogeneous tumours. Susceptible genetic background, accumulation of genetic changes, and environmental factors play an important role in gastric carcinogenesis. Single nucleotide polymorphisms (SNPs) in mitotic segregation genes could be responsible for inducing the slow process of accumulation of genetic changes, leading to genome instability. Patients and methods We performed a case-control study of polymorphisms in mitotic kinases TTK rs151658 and BUB1B rs1031963 and rs1801376 to assess their effects on gastric cancer risk. We examined the TTK abundance in gastric cancer tissues using immunoblot analysis. Results C/G genotype of rs151658 was more frequent in patients with diffuse type of gastric cancer and G/G genotype was more common in intestinal types of gastric cancers (p = 0.049). Polymorphic genotype A/A of rs1801376 was associated with higher risk for developing diffuse type of gastric cancer in female population (p = 0.007), whereas A/A frequencies were increased in male patients with subserosa tumour cell infiltration (p = 0.009). T/T genotype of rs1031963 was associated with well differentiated tumours (p = 0.035). TT+CT genotypes of rs1031963 and GG+AG genotypes of rs1801376 were significantly associated with gastric cancer risk (dominant model; OR = 2,929, 95% CI: 1.281-6.700; p = 0.017 and dominant model; OR = 0,364, 95% CI: 0.192-0.691; p = 0.003 respectively). Conclusions Our results suggest that polymorphisms in mitotic kinases TTK and BUB1B may contribute to gastric tumorigenesis and risk of tumour development. Further investigations on large populations and populations of different ethnicity are needed to determine their clinical utility.