ABSTRACT: BACKGROUND: Horizontal gene transfer (HGT) is considered to be a major force driving the evolutionary history ofprokaryotes. HGT is widespread in prokaryotes, contributing to the genomic repertoire of prokaryoticorganisms, and is particularly apparent in Rickettsiales genomes. Gene gains from both distantly and closelyrelated organisms play crucial roles in the evolution of bacterial genomes. In this work, we focus on genestransferred from distantly related species into Rickettsiales species. RESULTS: We developed an automated approach for the detection of HGT from other organisms (excludingalphaproteobacteria) into Rickettsiales genomes. Our systematic approach consisted of several specializedfeatures including the application of a parsimony method for inferring phyletic patterns followed by blast filter,automated phylogenetic reconstruction and the application of patterns for HGT detection. We identified 42instances of HGT in 31 complete Rickettsiales genomes, of which 38 were previously unidentified instances ofHGT from Anaplasma, Wolbachia, Candidatus Pelagibacter ubique and Rickettsia genomes. Additionally,putative cases with no phylogenetic support were assigned gene ontology terms. Overall, these transfers couldbe characterized as 'rhizome-like'. CONCLUSIONS: Our analysis provides a comprehensive, systematic approach for the automated detection of HGTs fromseveral complete proteome sequences that can be applied to detect instances of HGT within other genomes ofinterest.