Mycoplasma bovis is an important cause of bovine respiratory disease (BRD) in newly received cattle at fattening operations. However, little information on its within-pen transmission dynamics during a BRD outbreak is available. Such information is nevertheless crucial to adapt control measures during M. bovis-associated BRD outbreaks. The objective of the current study was to determine whether single or multiple clones of M. bovis are present within a pen during a BRD outbreak that occurs early in the feeding period. Sixteen BRD outbreaks that naturally occurred in 12 pens of 8-12 bulls each (n = 112) newly received at 3 fattening operations were investigated. Two hundred and thirty-nine transtracheal aspirations (TTA) were performed during the outbreaks, and the M. bovis isolates obtained were characterized by pulsed-field gel electrophoresis (PFGE). Mycoplasma bovis isolates were recovered from TTA in 8 of the 16 BRD outbreaks that occurred. The within-pen prevalence of bulls positive for M. bovis during these outbreaks ranged from 8% to 100%. The PFGE analysis revealed that, even though bulls came from multiple origins, a single clone of M. bovis was present within a pen during BRD outbreaks with a high prevalence of M. bovis infection. The study therefore indicates that, even if M. bovis can recrudesce from carriers after stressful events such as transportation and commingling, the increased prevalence of M. bovis pulmonary infection observed during BRD outbreaks that are early occurring in the feeding period seems primarily due to the horizontal transmission of only 1 clone among cattle.