Background Viral-associated trichodysplasia of immunosuppression is a rare cutaneous eruption that is characterized by follicularly based shiny papules and alopecia with characteristic histopathologic findings of abnormally anagen follicules with excessive inner root sheath differentiation. Prior reports have described the histopathologic characteristics on vertical sections; however, to our knowledge, immunohistochemical analysis of polyomavirus proteins has not been previously performed. Observations We discuss the thorough diagnostic evaluation and therapy of an unusual case of viral-associated trichodysplasia due to a newly described human polyomavirus that occurred in a patient with post-treatment chronic lymphocytic leukemia and an abnormal white blood cell count. Unique to our study is the immunohistochemical staining for the polyomavirus middle T antigen, which demonstrated positive staining of cellular inclusions within keratinocytes that compose the inner root sheath. Further evaluation with scanning electron microscopy and polymerase chain reaction analysis of viral DNA confirmed the presence of the virus. Treatment with topical cidofovir resulted in dramatic clinical improvement and hair regrowth. Conclusions Several tools, including immunohistochemical staining for the polyomavirus middle T antigen, can be used to identify the pathogenic virus associated with viral-associated trichodysplasia. This case highlights the utility of multiple diagnostic modalities and a robust response to a topical therapeutic agent, cidofovir.