Conservation trans-locations using captive bred red squirrels (Sciurus vulgaris) is increasing in the United Kingdom (UK). However, project managers are often unaware of the risk of pathological adenovirus (ADV) infection. In this study we illuminate the viral threat using transmission electron microscopy (TEM) and polymerase chain reaction (PCR) assays. Both techniques were used to screen samples collected from 26 English and Welsh captive red squirrel collections. Of 181 carcasses received between 2002 and 2016, 129 (71%) were suitable for routine surveillance post mortem examination (PME). A range of tissues were examined with ADV identified from a variety of samples by PCR and TEM in 92 (72%) cases encompassing 23 of the 26 study collections (89%). ADV enteritis was histologically confirmed in two deaths (2%) with another 39 (30%) through both laboratory and clinical findings, considered as likely clinically-significant ADV cases, but advanced autolysis precluded accurate assessment and confirmatory histological diagnosis. Other positive cases were more indicative of sub-clinical infection. Clusters of ADV red squirrel deaths were recorded with circumstantial evidence suggesting inter-collection movement of presumed ADV infected donated animals had triggered mortality in recipient collections. During the study, several collections intermittently experienced ADV-associated deaths. Definitive cause of death was not determined in most cases, but a diverse range of diagnoses were recorded in 25 (19%) animals. Implications of these findings for captive United Kingdom (UK) red squirrel husbandry are discussed. It is recommended that protocols be drawn up to minimise potential intra-species ADV infection and highlight the danger of contact with ADV infected wood mice (Apodemus sylvaticus).