An investigation was carried out in kharif 2010 to study morphological and molecular variation in Puccinia arachidis Speg., which causes rust of peanut. Five isolates were collected from different varieties grown in Main Agricultural Research Station (MARS ), Dharwad. Further, ten isolates were collected from different locations (North Karnataka; 7 isolates, Maharashtra; 2 isolates and Tamil Nadu; 1 isolate). Morphological variation was explored by studying various aspects of uredospore morphology viz., dimension, colour, shape, echinulations and spore surface. Isozyme variation was studied by using Polyacrylimid Gel Electrophoresis (PAGE) techniques. Selected isozymes viz., Peroxidase and Polyphenol Oxidase were studied to bring out variation. The uredospore morphology studies revealed that the dimension of spores of all isolates was in the range of 21-26 × 18-21 µm. Nearly all spores had light cinnamon colour and globose to ovoid shape. Within Dharwad location, the uredospores isolated from JL-24 were found to have the smallest length as well as width. Uredospores isolated from Annigere were found to be the smallest. Uredospores collected from Jalgaon were more globose and the spores collected from Coimbatore were more elliptical. The pits on the surface of the spores were observed in Annigere, Bellary, Jalgaon and Kolhapur isolates only. The echinulations were scarce to fine in all the isolates. A teleospore measuring 30.64 x 19.33 µm was found in the isolate collected from Gadag. Isozyme variation showed GPBD-4 and TAG-24 isolates were different from other isolates that were collected from different varieties within Dharwad location. Jalgaon and Gadag isolates always differed from the rest. In PAGE analysis of Peroxidase, cent per cent similarity was observed in all the isolates of Dharwad except that isolated from TAG-24. Comparatevely, maximum similarity (100 %) was noticed between Annigere and Bellary isolates. Polyphenol oxidase analysis, GPBD-4 and TAG-24 isolates showed maximum (100%) similarity. Location wise cent per cent similarity was noticed between Dharwad-Gadag, Dharwad-Kolhapur, and Gadag-Kolhapur isolates.