The file “Fla.fasta”, comprising 10526 positions, is the concatenated amino acid alignments of 51 orthologues of 182 bacterial strains. It was used for the maximum likelihood and maximum parsimony analyses of Flavobacteriales. Bacterial species names and strains were used as the sequence names, host names of insect endosymbionts were shown in brackets. The file “16S.fasta” is the alignment of 233 bacterial 16S rRNA sequences. It contains 1455 positions and was used for the maximum likelihood analysis of flavobacterial insect endosymbionts. The names of endosymbiont strains were replaced by the name of their hosts. In addition to the species names, National Center for Biotechnology Information (NCBI) accession numbers were also indicated in the sequence names (e.g., sequence “Cicadellidae_Deltocephalinae_Macrostelini_Macrosteles_striifrons_AB795320” is the 16S rRNA of Macrosteles striifrons (Cicadellidae: Deltocephalinae: Macrostelini) with a NCBI accession number AB795320). The file “Sulcia_pep.fasta” is the concatenated amino acid alignments of 131 orthologues of “Candidatus Sulcia muelleri” (Sulcia). It contains 41970 positions and presents 101 Sulcia strains and 3 Blattabacterium strains. This file was used for the maximum likelihood analysis of Sulcia. The file “Sulcia_nucleotide.fasta” is the concatenated nucleotide alignment corresponding to the sequences in “Sulcia_pep.fasta” but also comprises the alignment of 16S rRNA. It has 127339 positions and was used for the maximum likelihood and maximum parsimony analyses of Sulcia. Individual gene alignments (16S rRNA and 131 orthologues of Sulcia and Blattabacterium) are deposited in the compressed file “individual_gene_alignments.zip”, which were used to construct gene trees for multispecies coalescent analysis. The names of Sulcia strains were replaced by the name of their hosts in “Sulcia_pep.fasta”, “Sulcia_nucleotide.fasta” and the files in “individual_gene_alignments.zip”. In all the alignment files, gaps are indicated by “-”.