Canine parvovirus (CPV-2) modified-live virus vaccine strain can replicate in lymphoid tissues and intestinal mucosa after administration, being shed through canine faeces. Detection of vaccine strains has been reported in the bloodstream and faeces, potentially interfering with molecular diagnostic tests. The persistence of these strains in canine tissues has not yet been described. With this aim, canine tissues were tested during a molecular survey to screen for the presence of canine enteric viruses. Tissue samples from 165 dead dogs were tested by a conventional PCR assay. Positive samples and five commercial vaccines were subjected to sequence analysis. Vaccinal strains were detected and virus load was measured by using a set of real-time PCR assays using minor-groove binder (MGB) probes. Seventy-five dogs (45.4%) tested positive for CPV-2. Strains from 70 dogs were characterised as field variants. The presence of CPV sequences of vaccine origin was observed in the spleen, intestine, and mesenteric lymph nodes of five young dogs. Vaccinal strains were detected from 12 to 24 days after the last vaccine administration. Viral loads comprised between 6.3 × 10 and 9.95 × 10 DNA copies/10 µl of template. This study confirms that CPV vaccinal strains can be detected in canine tissues after vaccination, so post-mortem diagnosis of CPV infection needs further molecular analyses to assess the viral type (vaccine or field strains). The present study updates the current information on the persistence of CPV vaccine strains in canine tissues and their possible interference with molecular assays.