Technical Abstract: Small open reading frame (smorf) genes comprise the second largest Babesia bovis multigene family. All known 44 variant smorf genes are located in close chromosomal proximity to ves1 genes, which encode proteins that mediate cytoadhesion and contribute to immune evasion. In this study, we characterized the general topology of smorf genes, and investigated the gene repertoire, transcriptional profile, and SMORF expression in two distinct strains, T2Bo and Mo7. Sequence analysis using degenerate primers identified additional smorf genes in each strain, and demonstrated that the smorf gene repertoire varies between strains, with conserved and unique genes in both. Smorf genes have multiple semi- conserved and variable blocks, and a large hypervariable insertion in 20 of the 44 genes defines two major branches of the family, termed smorf A and smorf B. A total of 32 smorf genes are simultaneously transcribed in brain sequestered T2Bo strain B. bovis. Peptide- specific antiserum bound in immunoblots to multiple SMORF proteins with a range of sizes predicted by smorf genes, confirming translation of multiple smorf gene products from these transcripts. Antibody generated against a recombinant SMORF protein bound to the merozoites in a punctate pattern within the merozoite and in clusters in infected erythrocyte cytoplasm and membrane. However, surface exposed expression of SMORF epitopes similar to VESA proteins could not be demonstrated. These results indicate that the smorf multigene family is larger than previously described, and demonstrate that smorf genes are expressed and are undergoing variation, both within strains and in a lineage specific pattern independent of strain specificity. The function of these novel proteins is unknown.