The capacity of Mycoplasma genitalium to develop resistance to macrolides makes detection of macrolide resistance genes by rapid real-time PCR assays increasingly necessary in clinical diagnostic laboratories so as to initiate appropriate treatment as rapidly as possible. The aim of this retrospective and comparative study was to conduct the clinical evaluation of three commercially available kits for macrolide resistance detection. A total of 111 M. genitalium positive samples analyzed in the Clinical Microbiology Laboratory of the Miguel Servet University Hospital, Zaragoza (Spain) were used. After M. genitalium molecular confirmation, the three assays under study were evaluated and discrepant results were resolved via sequencing. The clinical sensitivity for resistance detection was 83% (95% confidence interval, 69% to 93%) for the ResistancePlussup.® MG panel kit (SpeeDx Pty Ltd., Sydney, Australia), 95% (84% to 99%) for Allplexsup.TM MG & AziR Assay (Seegenesup.®, Seoul, Korea), and 97% (88% to 99%) for the VIASURE macrolide resistance-associated mutations (23SrRNA) Real time PCR detection kit (Certest Biotec, Zaragoza, Spain). The clinical specificity was 100% (94% to 100%) for Allplex and VIASURE assays and 95% (86% to 99%) for SpeeDx assay. The results arising from this study are cause for strong consideration for the implementation of rapid real-time PCR assays in clinical diagnosis laboratories to eliminate treatment failure and transmission as soon as possible.