Chlorophyll is necessary for photosynthesis in plant and affecting for crop yield. Chlorophyll content can be directly measured, however, that method is destructive leaves. In sugarcane, indirect methods were used for drought or stress condition however literature is lacking for normal condition. The objective was to study the relationship between direct and indirect chlorophyll measurement. The experiment was conducted under 2 locations in Mahasarakham province, Thailand. RCBD with 4 replications and 16 varieties were used. The chlorophyll content (CC), SPAD chlorophyll meter reading (SCMR) and chlorophyll fluorescence (CF) were measured five times and a month interval between 8-12 months after planting. The results revealed that CC and SCMR of 16 varieties in both locations were significantly different, however, CF was not significantly different. The interaction between location and varieties were found. The relationships between methods of measurement were also found. At Kut Rung district, CC and SCMR were positively correlated in 8 to 12 months except for 11 months (0.71**, 0.55*, 0.77**and 0.78** respectively). Another location, CC and SCMR were correlated in 8 and 9 months (0.51*and 0.73** respectively) and CF was not correlated with CC and SCMR. Combined analysis, CC was positively correlated with SCMR at 8- 12 months except for 11 months (0.61*, 0.55*, 0.63**and 0.50* respectively). SCMR is a useful strategy for indirectly chlorophyll measurement in sugarcane. CF could not apply for measurement in this case. For further research, we are looking for a relationship between chlorophyll content and sugarcane yield or other desirable characteristics in sugarcane.