Gluconacetobacter diazotrophicus is an endophytic diazotroph in most of the monocot plants and in sugar rich crops. The present study attempts to isolate and characterize the Gluconacetobacter diazotrophicus which was isolated from the tissues of surface sterilized roots of four different crops viz., sugarcane, maize, tomato and bajra and from cane juice of sugarcane. Forty isolates were subjected for morphological, cultural and biochemical tests for the characterization. The endophytic isolates obtained from different crops showed phenotypic and biochemical characteristics identical to those internationally reported in Bergey’s Manual of Systemic Bacteriology (2005).