In dogs with diet-associated dilated cardiomyopathy (DCM), we have identified electron microscopic changes suggestive of abnormal lysosomal accumulation of phospholipids and consistent with the appearance of drug-induced phospholipidosis in people and other animals. The objective of this study was to compare concentrations of urine di-docosahexaenoyl (22:6)-bis(monoacylglycerol)phosphate (BMP), a biomarker of drug-induced phospholipidosis, in dogs with DCM eating high-pulse (HP) diets, dogs with DCM eating low-pulse (LP) diets, and healthy controls (control-HP and control-LP). In this cross-sectional study, voided urine was collected from client-owned dogs with DCM from September 2018 through March 2020. Urine di-22:6-BMP was measured by LC-MS-MS and normalized to urine creatinine. Normalized di-22:6-BMP concentrations were compared among groups using mixed-effects-model analysis. 53 dogs were included: DCM-HP (n = 25), DCM-LP (n = 4), control-HP (n = 10), and control-LP (n = 14). Mixed-effects models adjusted for age and sex showed that HP diet was significantly associated with higher normalized urine di-22:6-BMP concentrations. A 1-way ANOVA identified a significant difference among the 4 groups, with Tukey post hoc analysis showing that the DCM-HP group had significantly higher normalized urine di-22:6-BMP concentrations compared to the control-LP group. Normalized di-22:6-BMP concentrations were significantly positively correlated with diet pulse scores (r = 0.52). High-pulse diets were significantly associated with higher normalized urine di-22:6-BMP concentrations. These results support the possible presence of primary or secondary phospholipidosis in dogs with diet-associated DCM and provide a plausible mechanism for further investigation.