Colletotrichum gloeosporioides (Penz.) Penz and Sacc, causal organism of anthracnose disease in pomegranate, sweet orange, banana and guava. The isolates of C. gloeosporioides designated as CgPD 1, CgPJ 2, CgPN 3, CgSD 4, CgSJ 5, CgSN 6, CgBD 7, CgBJ 8, CgBN 9, CgGD 10, CgGJ 11 and CgGN 12. The diseased sample collected from four different fruit crops and three different district in Khandesh region of Maharashtra. These isolates were characterized for cultural variation. The culture of isolate CgPD 1 recordrd maximum average growth rate of 12.85 mm/ day while, isolate CgBN 9 recorded lowest growth with 9.15 mm/ day. Most of the isolates shows excellent sporulation. The colony colour of isolate shows white, pinkish, creamy white and white to dull white. Most of the isolates showed raised fluffy, regular smooth growth. The pigmentation varied from white, pinkish and white to orange. Among the five culture media (one synthetic and four non synthetic) media tried, the maximum growth of C. gloeosporioides irrespective of isolates was obtained on Potato Dextrose Agar (7.48 mm day·l). Host Leaf Extract Agar and Oat Meal Agar medium were also statistically significant followed by with Potato Dextrose Agar and recorded average growth rate of 5.98 mm /day and 5.19 mm /day, respectively. The Peptone Rose Bengal Agar medium was found in poor growth of isolates (3.77 mm/day).