Canine bocaviruses (CBoVs) have been recognized as pathogens associated with intestinal diseases. Hematogenous spreading caused by CBoV has been documented and may potentiate the virus entry across the blood-brain barrier to initiate a brain infection. This study focused attention on CBoV detection in cases of encepahlopathy and attempted to determine its viral localization. A total of 107 dog brains that histologically exhibited encephalopathy (ED) were investigated for the presence of CBoVs using polymerase chain reaction (PCR). Thirty-three histologically normal brain samples from dogs were used as a control group (CD). CBoV-2 was detected in 15 ED dogs (14.02%) but not in CD dogs (p = 0.02), while no CBoV-1 and -3 were detected. Among the CBoV-2 positive dogs, brain histological changes were characterized by nonsuppurative encephalitis, with inclusion body-like materials in some brains. In situ hybridization (ISH) and transmission electron microscopy (TEM) confirmed the presence of CBoV-2 viral particles in glial cells, supporting neurotropism of this virus. ISH signals were also detected in the intestines, lymphoid organs, and the heart, suggesting both enteral and parenteral infections of this virus. Whole genome characterization and evolutionary analysis revealed genetic diversity of CBoV-2 sequences and it was varying among the different countries where the virus was detected. This study points to a possible association of CBoV-2 with encephalopathy in dogs. It also highlights the genetic diversity and cellular tropism of this virus.