The unexpected seroconversion of sentinel mice in our facility to murine T lymphotrophic virus (MTLV) positivity led to our identification of a novel murine astrovirus that we designated murine astrovirus 2 (MuAstV-2). During our investigation, MuAstV-2 was found to be a contaminant of the T helper cell line (D10. G4.1) that was used to generate the MTLV antigen that we included in the multiplex fluorometric immunoassay (MFIA) that we used for sentinel screening. We eventually determined that cross-reactivity with the astrovirus generated a positive result in the MTLV assay. A confirmatory immunofluorometric assay (IFA) using the same MTLV-infected cell line yielded a similar result. However, the use of antigen prepared from MTLV-infected neonatal mouse thymus did not reproduce a positive result, leading us to suspect that the seroreactivity we had observed was not due to infection with MTLV. A mouse antibody production test showed that mice inoculated with naïve D10. G4.1 cells and their contact sentinels tested positive for MTLV using cell-line generated antigen, but tested negative in assays using MTLV antigen produced in mice. Metagenomic analysis was subsequently used to identify MuAstV-2 in feces from 2 sentinel mice that had recently seroconverted to MTLV. Two closely related astrovirus sequences (99.6% capsid identity) were obtained and shared 95% capsid amino acid identity with the MuAstV-2 virus sequenced from the D10. G4.1 cell line. These viruses are highly divergent from previously identified murine astroviruses, displaying <30% capsid identity, yet were closely related to murine astrovirus 2 (85% capsid identity), which had recently been isolated from feral mice in New York City. A MuAstV-2 specific PCR assay was developed and used to eradicate MuAstV-2 from the infected colony using a test and cull strategy. The newly identified MuAstV2 readily transmits to immunocompetent mouse strains by fecal-oral exposure, but fails to infect NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, which have significantly impaired adaptive and innate immune systems. Neither immunocompetent nor immunodeficient mice showed any astrovirus-associated pathology. MuAstV-2 may provide a valuable model for the study of specific aspects of astrovirus pathogenesis and virus-host interactions.