Questions about pathogenesis and therapy for Helicobacter infections in dogs could be answered with a simple, noninvasive, sensitive, and specific diagnostic test. We hypothesized that a fecal polymerase chain reaction (PCR) assay would detect Helicobacter and could be useful for assessing therapeutic responses. Paired gastric biopsies and fecal samples were obtained from 39 random source dogs (group 1). Gastric biopsies from each of these dogs had histologic evidence of gastric spiral bacteria, and paired gastric tissue and fecal samples produced a 375-base pair (bp) product when amplified by PCR with Helicobacter-specific primers. Specificity of the PCR product was confirmed by detection of expected 60-, 119-, and 196-bp products following Hinfl digestion. Direct sequencing of amplicons from paired PCR products from gastric biopsy and fecal samples from 8 group I dogs showed that gastric products had the highest homologies with known gastric Helicobacter species, whereas fecal products had the highest homologies with intestinal species. Healthy mixed-breed dogs (group II; n = 8) with histologically confirmed spiral bacteria infection were treated with a 21-day course of metronidazole, amoxicillin, and famotidine. Fecal samples were collected from group II dogs twice before and within 3 days of completion of treatment. The PCR results correctly identified 15/16 pretreatment samples as positive: 1 pretreatment sample was negative. PCR results identified 8/8 posttreatment samples as Helicobacter negative. Fecal PCR is a useful test for detecting Helicobacter infection in dogs. This assay may be useful as a screening test for infection and could be used to address questions relevant to pathogenesis and therapy.