PURPOSE: To investigate the diagnostic performance of multicolor imaging for detection of retinal nerve fiber layer (RNFL) defects in myopic eyes with glaucoma. DESIGN: Comparative diagnostic analysis. METHODS: A total of 150 myopic eyes of 138 patients (< -1.00 diopter or axial length >24 mm) underwent multicolor imaging by Spectralis (Heidelberg Engineering GmbH) optical coherence tomography (OCT). Two independent observers graded the RNFL visibility score and determined the location of the RNFL defect on each image. Readings were taken from the superior and inferior areas. The diagnostic performances of multicolor, green-reflectance, and blue-reflectance images for detection of the RNFL defect were compared with those of conventional fundus and red-free RNFL photographs. RESULTS: The interobserver agreement on the RNFL visibility score was substantial for fundus and RNFL photographs and multicolor and green-reflectance images and was perfect for blue-reflectance images. The interobserver agreement on the location of the RNFL defect was better when using multicolor imaging than when using conventional photography. The green-reflectance image (2.89 +/- 0.31) showed the highest RNFL visibility score, followed by blue-reflectance (2.88 +/- 0.32) and multicolor (2.57 +/- 0.63) images. The sensitivity of multicolor imaging (94.9%, 93.2%, and 91.5% for green-reflectance, blue-reflectance, and multicolor images, respectively) was significantly higher than that of fundus photography (83.1%) for detection of superotemporal RNFL defects (all P < .05). It was significantly higher for inferotemporal defects than those of fundus and RNFL photographs (all P < .05). CONCLUSIONS: Multicolor imaging can visualize the glaucomatous RNFL defects in myopic eyes that are indistinguishable on conventional photography.