Background: The objective of the present study was to compare, utilising two guideline assessment instruments, six corresponding clinical practice guidelines of the British Association for Sexual Health and HIV and the Centres for Disease Control. Methods: Three raters independently assessed the recently published guidelines for gonorrhoea, chlamydial infection, early syphilis, pelvic inflammatory disease, bacterial vaginosis and HIV testing using two instruments, the Cluzeau and the AGREE (Appraisal of Guidelines for Research and Evaluation instrument). The Cluzeau scores were a simple percentage comparison; the AGREE scores were a standardised score for each guideline development domain. Differences were assessed using the Wilcoxson signed ranks test. Inter-rater variability was calculated on the Cluzeau instrument utilising the intragroup correlation method. Results: The British Association for Sexual Health and HIV guidelines scored higher than the Centres for Disease Control guidelines in many of the assessed domains. There were significant differences between the two in many of the scores (P = 0.026–0.028). Inter-rater concordance was high to very high at 0.70–0.83. Conclusions: There were often major differences in scores between the two guideline groups. It is necessary for wider discussion within the profession to consider the significance of these findings.