Neisseria gonorrhoeae engages in extensive intra-cellular gene conversion between the PilE-expression locus (pilE) and the transcriptionally-silent pil gene copies (pilS). In silico analyses were applied to investigate the extent of sequence heterogeneity between the various pilS gene copies. Analysis of synonymous and non-synonymous substitutions between the different pilS genes indicated that relatively few amino acid changes would occur due to nucleotide polymorphisms towards the 5’ end of the pilS genes whereas more frequent amino acid substitutions would be incorporated within the “hypervariable” region. The lack of non-synonymous substitutions at the 5’ end of the genes was found to be under selective pressure as indicated by a positive D_T score utilizing the Tajima test. The presence or absence of mismatch repair appeared to only impact recombination when non-identical DNAs recombined via the DNA transformation route, where small pil sequence heterogeneities were sufficient to terminate recombination tracts, with these sequence constraints being relieved in cells carrying a mutS mutation. Therefore, the data indicate that the effect of sequence heterogeneity on recombination within the pil system appears to depend upon the context with which the non-identical DNAs recombine.