This study was carried out to deduce an appropriate analysis method to evaluate the homogeneity of the agricultural characteristics of genetically modified (GM) crops for environmental risk assessment. Comparative analyses were made between GM hot pepper lines and their control lines cultivated in the same year, between different GM peppers carrying the same gene but in different chromosomal loci, between the same GM peppers cultivated in different years and two different GM peppers carrying different genes from three GM peppers (B20, H15, and T20) and their control lines (P915, P2377, and THK). The aim was to determine the most appropriate comparison combination to establish the evaluating protocol and environmental risk assessment. Data of 57 different characteristics of pepper plants, green fruits and red fruits was collected during the optimal vegetation period. Of the 57 different characteristics, 24 characteristics were excluded as there was no difference and the remaining 33 characteristics were analyzed by t-test. There were statistical differences in five characteristics between the B20 and P915 lines, five characteristics between the H15 and P2377 lines and two characteristics between the T20 and THK lines cultivated either in 2008 or in 2011. On the other hand, there were significant differences in 11 characteristics between the B20 and H15 lines cultivated in 2008 and 14 characteristics between the H15 and T20 harvested in 2011. Comparative analysis of 33 agricultural characteristics between the H15 line cultivated in 2008 and 2011 showed the most significant differences. Based on this result, the comparison between the same GM lines cultivated in different years was judged meaningless when the homogeneity of agricultural characteristics of GM crops were evaluated. In conclusion, the comparative analysis between GM peppers and their control lines cultivated in the same year is the most appropriate while the homogeneity of agricultural characteristics is examined to assess environmental risk of GM peppers.