The assessment of the corneal nerve fibre plexus with corneal confocal microscopy (CCM) is an upcoming but still experimental method in the diagnosis of early stage diabetic peripheral neuropathy (DPN). Using an innovative imaging technique-Heidelberg Retina Tomograph equipped with the Rostock Cornea Module (HRT-RCM) and EyeGuidance module (EG)-we were able to look at greater areas of subbasal nerve plexus (SNP) in order to increase the diagnostic accuracy. The aim of our study was to evaluate the usefulness of EG instead of single image analysis in diagnosis of early stage DPN. This prospective study was performed on 60 patients with type2 diabetes mellitus, classified equally into two subgroups based on neuropathy deficient score (NDS): patients without DPN (group1) or with mild DPN (group2). The following parameters were analysed in the two subgroups: corneal nerve fibre length (CNFL; mm/mm2), corneal nerve fibre density (CNFD;no./mm2), corneal nerve branch density (CNBD;no./mm2). Furthermore, we compared the data calculated with the novel mosaic, EG-based method with those received from single image analysis using different quantification tools. Using EG we did not find a significant difference between group1 and group2: CNFL (16.81 ± 5.87mm/mm2 vs. 17.19 ± 7.19mm/mm2, p = 0.895), CNFD (254.05 ± 115.36no./mm2 vs. 265.91 ± 161.63no./mm2, p = 0.732) and CNBD (102.68 ± 62.28no./mm2 vs. 115.38 ± 96.91no./mm2, p = 0.541). No significant difference between the EG method of analysing the SNP and the single image analysis of 10 images per patient was detected. On the basis of our results it was not possible to differentiate between early stages of large nerve fibre DPN in patients with type2 diabetes mellitus via SNP analysis. To improve sensitivity and specificity of this method newer technologies are under current evaluation. ClinicalTrials.gov Identifier NCT05326958.