The positional isomers of tert-butylnitroxide (NO) substituted 4,5,9,10-tetramethoxypyrene-based mono- and biradical are synthesized. While the biradical 2,7-TMPNO in which two NO radical moieties are attached at the nodal plane of pyrene adopts a semiquinoid structure, the 1,6- and 1,8-isomers of the same exist in biradical form. The tuning of the antiferromagnetic exchange interactions is achieved by synthesizing the positional isomers of the biradical while maintaining the same radical moiety as well as the pi spacer.