Juvenile Oyster Disease (JOD) causes devastating losses to the Eastern oyster (Crassostrea virginica) industry, with mortality rates up to 100% in warmer temperatures. The primary causative agent is the bacterium Aliiroseovarius crassostreae. Current detection relies on traditional PCR, and the incomplete genome hinders discrimination of virulent A. crassostreae strains from environmental isolates. Here, we developed a duplex quantitative PCR (qPCR) assay targeting virulent A. crassostreae strains in Eastern oysters, validated according to Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, demonstrating an efficiency of 90-110%. Additionally, we assembled and annotated the complete genome of A. crassostreae (4.2 Mb) using Oxford Nanopore sequencing and performed phylogenomic analyses to clarify inter- and intra-species taxonomic relationships within the Roseobacter family. This integrated genomic and diagnostic approach enables rapid, specific detection of JOD, minimizing economic losses for oyster farmers.