Hepatitis B virus (HBV) is a human pathogen of global concern, while a high diversity of viruses related to HBV have been discovered in other animals during the last decade. Recently, the novel mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), was detected in an immunocompromised cat. Herein, a collection of 209 cat sera and 15 hepato-diseased cats were screened for DCH using PCR, resulting in 12.4% and 20% positivity in the tested sera and necropsied cats, respectively. Among the DCH-positive sera, a significantly high level of co-detection with retroviral infection was found, with the highest proportion being co-detection with feline immunodeficiency virus (FIV). Full-length genome characterization of DCH revealed the genetic diversity between the nine Thai DCH sequences obtained, and that they phylogenetically formed three distinct monophyletic clades. A putative DCH recombinant strain was found, suggesting a possible role of recombination in DCH evolution. Additionally, quantitative PCR was used to determine the viral copy number in various organs of the DCH-moribund cats, while the pathological findings were compared to the viral localization in hepatocytes, adjacent to areas of hepatic fibrosis, by immunohistochemical (IHC) and western blot analysis. In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, providing evidence of systemic infection. Ultrastructure of infected cells revealed electron-dense particles in the nucleus and cytoplasm of hepatocytes, bronchial epithelial cells, and fibroblasts. We propose the intracellular development mechanism of this virus. Although the definitive roles of pathogenicity of DCH remains undetermined, a contributory role of the virus associated with systemic diseases is possible.