The heterotopic submucosal gland (HSG) is a common incidental finding in gastrectomy specimens. The majority of HSGs are small incidental lesions, which are also known as gastritis cystica profunda. However, larger lesions may appear as an inverted growth of well-organized mucosa referred to as gastric inverted polyps.To determine whether genetic alterations are involved in HSG development, we analyzed 63 gastric HSG lesions using targeted next-generation sequencing and immunohistochemistry.Histologically, HSG lesions consistently had areas of pyloric gland differentiation with variable extent of foveolar differentiation. Although the background mucosa showed intestinal metaplasia in most cases (98%), intestinal-type epithelium was seen in only one HSG lesion (2%). Sequencing analysis identified activating KRAS, BRAF, CTNNB1, and GNAS mutations in 34 (54%), 1 (2%), 1 (2%), and 7 (11%) lesions, respectively. HSG lesions harboring a KRAS mutation were more likely to present extensive foveolar differentiation (P = 0.013) and absence of parietal cells (P = 0.0081). Five HSG lesions had a dysplastic component, and concordant genetic alterations were detected between the non-dysplastic and dysplastic areas of two lesions that were successfully analyzed. Immunohistochemical staining demonstrated diffuse expression of mutant KRAS protein in lesions with the most common genetic alteration, KRAS G12D.Our study demonstrated that a major proportion of HSGs were proliferative lesions associated with oncogenic mutations, with more than half of lesions harboring activating KRAS mutations.