Tylosin is a veterinary antibiotic and is commercially produced using Streptomyces fradiae. Previously, we developed a mutant gamma-1 of S. fradiae NRRL-2702 with a 6.87-fold increase in tylosin yield as compared with the wild-type strain through irradiation mutagenesis. The present studies were conducted to explore mutational changes in regulatory genes (TylQ, TylP, TylS, TylR, and TylT) of Tyl cluster that may lead to an enhanced expression of tylosin. Expression analysis by RT-PCR revealed that TylQ was switched off earlier in mutant gamma-1 while no change in expression pattern of TylP was observed between the wild-type and mutant gamma-1 strains. However, a point mutation with a substitution of T to A was recorded at position 214 in the 420-bp product of TylP from mutant gamma-1 that resulted in a change of one amino acid (serine to threonine) at position 72. Moreover, no mutation in the nucleotide sequence of TylS, TylR, and TylT genes was detected.