Xylella fastidiosa Strain Stag’s Leap, originally isolated from Napa Valley, California, is highly virulent in causing Pierce’s Disease (PD) of grapevine. Plasmids are extrachromosomal genetic elements associated with bacterial environmental adaptation such as virulence development. In this study, the plasmid status of Strain Stag’s Leap was studied. DNA samples extracted from pure culture in PW medium (in vitro) and PD grapevine in greenhouse (in planta) were subject to next generation sequencing (NGS, MiSeq/HiSeq formats). Through de novo assembling and primer walking techniques, a circular plasmid, pXFSL21, of 21,665 bp was identified from MiSeq data (in vitro DNA) and confirmed by HiSeq data (in planta DNA) using referenced assembling. NGS read mapping and PCR experiments indicated that pXFSL21 was single-copied per genome both in vitro and in planta. The plasmid had 27 genes/open reading frames (ORFs) encoding proteins of DNA replication (DR), conjugal transfer (CT), two toxin/antidote (TA) systems, and a multidrug resistance (MDR) efflux pump. BLAST search against current GenBank database (version 222.0) showed that pXFSL21 was unique for its TA and MDR genes among the 46 deposited X. fastidiosa plasmids. However, among the whole genome sequences of 41 X. fastidiosa strains, MDR homologs were found in 29 strains and TA system genes were found in four strains. Phylogenetic analyses suggested genes of DR, CT, TA, and MDR were likely of different origins.