How virulent protozoal pathogens capable of causing overt disease are maintained in nature is an important paradigm of eukaryotic pathogenesis. Here we used population genetics and molecular methods to study the evolution and emergence of a marine invasion of new genetic variants of Toxoplasma gondii, referred collectively as Type X (HG12). 53 Toxoplasma isolates were obtained from mustelids that stranded between 1998-2004 with toxoplasmosis (ranging from chronic infection to fatal encephalitis). Over 74% of the sea otters collected throughout their geographic range were infected with Type X as determined by multi-locus PCR-DNA sequencing. Depending on the locus investigated, Type X strains possessed one of three allelic types that had independently assorted across the strains examined either genetically distinct alleles, referred to as “gamma” or “delta”, or a Type II allele. Phylogenetic incongruence among locus-specific trees, genome-wide CGH array and WGS analyses confirmed that Type X is a sexual clade of natural recombinants that resemble F1 progeny from a genetic cross between Type II and a mosaic of two distinct “gamma” or “delta” 28 ancestries. A single Type X genotype (19/53 36%) had expanded in sea otters largely as subclinical chronic infections, but it was highly pathogenic to mice (LD100= 1 parasite). To determine whether murine virulence genes could be mapped within this naturally occurring population, we performed a genome scan and identified four QTLs with LOD scores greater than 4.0. Targeted disruption of ROP33, the strongest candidate from among 16 genes within the highest QTL on Chromosome VIIa established ROP33 as a murine virulence locus. The ability of this highly pathogenic clone to expand and cause the majority of sea otter infections supports a virulence shift model whereby generalist pathogens like Toxoplasma utilize their sexual cycles to produce new strains with an expanded biological potential. Such a trait enables pathogens to extend their host range or be naturally selected within their vast intermediate host range to maximize their transmission. Our work thus establishes a rationale for how virulent strains can be maintained cryptically in nature across a pathogen’s broad host range, and act as reservoirs for epidemic disease. Contact: Michael E Grigg. This submission was powered by METAGENOTE (https://metagenote.niaid.nih.gov).