Shiga toxin (Stx)-producing Escherichia coli strains are foodborne pathogens that can cause severe human diseases, such as haemorrhagic colitis and haemolytic-uraemic syndrome. Stxs are encoded by bacteriophages (Stx phages) which show remarkable variations in genome composition and harbour several genes of unknown function. Recently, a gene encoding a sialate O-acetylesterase (NanS-p) was identified in some relevant Stx2a phages and it was suggested that it could provide advantages for bacterial growth in the gut. The aim of this study was to analyze the presence and sequence of nanS-p genes in available Stx2a genomes. A total of 59 DNA sequences of Stx2a phages were extracted from NCBI GenBank database with the BLASTN program using the stx_2a sequence from the phage 933W as query sequence, either as complete phage genomes (45) or from bacterial genomes by subsequent analysis with PHASTER web server (14). Comparative analysis revealed that nanS-p was located downsteam stx_2a in all genomes. Twenty different amino acid sequences of NanS-p were identified among the 59 Stx2a phages. Specifically, catalytic esterase domains were clustered in 11 groups, with differences mainly observed in nine amino acid positions. Sequences corresponding to the N-terminal domain (DUF1737) clustered into three groups, two of them closely related, while C-terminal domain was highly variable giving place to four groups. Since sialate O-acetylesterase activity has been determined from particular Stx2a phages, new studies are necessary to evaluate if the NanS-p subtypes identified in the present study also differ in their biological activity.