Plasmids are important genetic elements contributing to bacterial evolution and environmental adaptation. Xylella fastidiosa is a nutritionally fastidious Gram-negative bacterium causing economically devastating diseases such as Pierce's disease (PD) of grapevine. In this study, the plasmid status of a highly virulent PD strain, Stag's Leap, originally isolated from Napa Valley, CA, was studied using sequencing and bioinformatics tools. DNA samples extracted from a pure culture in periwinkle wilt medium (in vitro DNA) and a PD-symptomatic grapevine artificially inoculated in the greenhouse (in planta DNA) were subject to next-generation sequencing (NGS) analyses (Illumina MiSeq or HiSeq). Sequence analyses and polymerase chain reaction experiments revealed the presence of a circular plasmid, pXFSL21, of 21,665 bp. This plasmid existed as a single copy per bacterial genome under both in vitro and in planta conditions. Two toxin-antitoxin (T-A) systems (ydcD-ydcE and higB-higA) were detected in pXFSL21, a possible mechanism for the long-term survival of this single-copy plasmid in the bacterial population. BLAST searches against the GenBank database (version 222) detected homologs of the two T-A systems in chromosomes or plasmids of some X. fastidiosa strains. However, double T-A systems were found only in pXFSL21. pXFSL21 was not found in other known PD strains and, therefore, could serve as a molecular marker for strain Stag's Leap monitoring and tracking. The NGS-based technique outlined in this article provides an effective tool for identifying single-or low-copy-number plasmids in fastidious prokaryotes.