Three groups of Iris dichotoma with different flower colors were investigated by morphological comparison and Karyotype analysis. The results indicated that there were obvious differences among the three groups with violet, yellow and white flowers, respectively. Their flowering time were 15:00, 14:00 and 16:00, and closing time were was 19:30, 19:00 and 20:00, respectively. The respective average variation coefficients of I. dichotoma with different flower colors were 10.47%~65.16%, 4.46%~45.95% and 3.95%~55.43%. The variation of 13 characters of three groups such as flower diameter, outer perianth, inner perianth and fruit length had significant difference, while there was no significant difference in the tillering number and the number of seeds. The analysis of karyotype demonstrated that the karyotype formula for I. dichotoma (Violet) was 2n=2x=32=12sm 20m, and the karyotype was 1B. The chromosome number and karyotype of I. dichotoma(Yellow) were reported here for the first time and its karyotype formula was 2n=2x=32=6sm 26m, belonging to "1A" type. The karyotype of I. dichotoma(White) was 2B, whose karyotype formula was 2n=2x=32=8sm 24m. By the analysis of karyotype and variation of morphological characters, the genetic relationships among the three different flower color groups of I. dichotoma were discussed, which showed that their phylogenetic relationships were relatively close and I. dichotoma(Yellow) as well as I. dichotoma(White) should be the intraspecific variation of I. dichotoma (Violet).