Sugarcane is a significant crop plant with the capability of accumulating higher amount of sucrose. In the present study, a high sucrose content sugarcane mutant clone, GXB9, has been studied in comparison to the low sucrose mother clone B9 on morphological, agronomical and physiological level in order to scrutinize the variation because of mutation in GXB9 in field under normal environmental condition. The results showed that GXB9 has less germination, tillering rate, stalk height, leaf length, leaf width, leaf area, number of internodes, internode length and internode diameter than B9. Qualitative traits of leaf and stalk displayed significant variation between GXB9 and B9. Endogenous hormones quantity was also showed variation between the two clones. The relative SPAD reading and chlorophyll a, b concentrations also showed variation between GXB9 and B9. The photosynthetic parameter analysis indicated that the GXB9 has significantly higher net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (Tr) than B9. The qRT-PCR analysis of genes encoding enzymes like SPS, SuSy, CWIN, and CeS showed upregulation in GXB9 and downregulation in B9. However, these genes were significantly differentially expressed between the immature and maturing internodes of GXB9. The cane quality trait analysis showed that GXB9 had higher juice rate, juice gravity purity, brix, juice sucrose content and cane sucrose content than B9. The yield and component investigation results indicated that GXB9 had lower single stalk weight, however higher number of millable stalks per hectare than B9, and GXB9 had lower theoretical cane yield than B9. SSR marker analysis showed genetic variation between GXB9 and B9. This study has shown significant variation in the traits of GXB9 in comparison to B9 which advocates that GXB9 is a high sugar mutant clone of B9 and an elite source for future breeding.