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Introduction Sexually transmitted infections (STIs) are common in our environment, and trends have been increasing in the last few years. Different methods for STIs diagnosis have been applied by microbiology laboratories over years, but real-time PCR has improved this process. Our objective was to evaluate VIASURE Sexually Transmitted Diseases Real-Time PCR Detection kit (CerTestBiotec, S.L.) comparing with the real-time PCR technique used in our laboratory (Allplex™ STI7 Essential Assay, Seegene) which was considered as reference assay. Methods A total of 948 samples from different sites (vaginal, endocervical, urethral, rectal, pharyngeal swabs and urine samples) were analyzed from July to September 2018. Results A discordant result was obtained in 4.5% (43 samples). These discrepancies were mainly observed in threshold cycle (Ct) value next to the limit of detection. The k coefficient obtained shows a very high agreement between both methods with k values from 0.92 to 0.99. Conclusions VIASURE Sexually Transmitted Diseases Real-Time PCR Detection kit provides a very good correlation with Allplex STI7 and therefore, it's a good tool for the diagnostic of STIs. Positive results with Ct value obtained from 35 and low amplification signal should be applied with caution and should be interpreted based on the patient's clinical data.