Degenerate oligonucleotide primers, designed based on conserved regions of several serine-threonine kinases (STK) previously cloned in tomato and Arabidopsis, were used to isolate STK candidates in wild and cultivated strawberries. Seven distinct classes of STKs were identified from three related wild species, i.e., Fragaria vesca, Fragaria chiloensis, and Potentilla tucumanensis, and seven different Fragaria x ananassa cultivars. Alignment of the deduced amino acid sequences and the Pto R protein from tomato revealed the presence of characteristic subdomains and conservation of the plant STK consensus and other residues that are crucial for Pto function. Based on identity scores and clustering in phylogenetic trees, five groups were recognized as Pto-like kinases. Strawberry Pto-like clones presented sequences that were clearly identified as the activation segments contained in the Pto, and some of them showed residues previously identified as being required for binding to AvrPto. Some of the non-Pto-like kinases presented a high degree of identity and grouped together with B-lectin receptor kinases that are also involved in disease resistance. Statistical studies carried out to evaluate departure from the neutral theory and nonsynonymous/synonymous substitutions suggest that the evolution of STK-encoding sequences in strawberries is subjected mainly to a purifying selection process. These results represent the first report of Pto-like STKs in strawberry.