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Helicobacter pylori is a well-established cause of gastritis and gastric malignancy, but other Helicobacter species—collectively termed non-Helicobacter pylori Helicobacter (NHPH)—also contribute to gastric disease. This study retrospectively analysed the prevalence of NHPH in 1115 routine gastric biopsies from a large academic medical centre submitted for H. pylori drug susceptibility genotyping using a next-generation sequencing (NGS) assay targeting 16S and 23S rRNA genes. NGS results of identified pathogens were compared against those identified on histology. NHPH species were detected in 15 of 1115 cases (1.3%), including 7 NHPH-only infections and 8 mixed infections with H. pylori. Detected NHPH species included Helicobacter heilmannii, Helicobacter felis and Helicobacter bizzozeronii. No mutations associated with antimicrobial resistance were identified in NHPH. Broader molecular testing may improve recognition of mixed infections and guide more accurate diagnosis and treatment for gastric disease.