Variation in the distribution and diversity of endophytic fungi associated with Calotropis gigantea with respect to ecological and seasonal conditions was assessed. It has been noticed that diversity of endophytic fungi and colonization frequency varied with the seasons with least in summer and highest in winter. A preference of colonization of different fungal species was also observed. It is also evident that diversity of endophytic fungi varied with the geographical distribution of the plant. Similarly, dominance of fungal species varied both with season and plant part. In conclusion it has been pointed out that more attention should be paid to investigating the endophytic fungal diversity in host plant growing under varied ecological and seasonal conditions which is more likely to reveal the behavior of endophytic fungi and the metabolites produced by them.