Skeletal muscle is composed of multinucleated muscle fibers, which play a crucial role in determining the quality of meat products in livestock. Quantifying the total number of muscle fibers (TNM) is essential for understanding muscle composition, but this remains challenging in poultry, particularly since the considerable number of livestock complicates the preparation of tissue sections for analysis and makes the counting process laborious. Our previous study developed an automatic muscle fiber quantification tool powered by deep learning, named MyoV, which has addressed this bottleneck. This study employed the MyoV tool for accurately quantifying TNM in the pectoral muscles of slow-growing (SL), medium-growing (ML), and fast-growing (FL) broilers. The results showed that FL group exhibited higher growth performance compared to ML and SL groups from the embryonic to rearing stages. Processing of whole slide images of pectoral muscle revealed significantly higher TNM in FL and ML groups than in SL group (P<0.01). The TNM values of FL, ML and SL groups were 693,568.00±54,169.80, 652,122.00±65,822.60, and 539,778.57±40,722.94 at 7 days of age (D7), respectively; and 663,014.93±58,801.11, 645,784.76±80,204.34 and 507,280.29±98,092.16 at D35 for FL, ML and SL groups, respectively. Differences in the cross-sectional area (CSA) of muscle fibers among the three groups were consistent with the TNM results. A correlation analysis showed correlation coefficients of 0.73–0.89 between body weight (BW) and TNM and 0.78–0.87 between BW and CSA. These findings directly indicate that the number of muscle fibers in broilers is an important foundation for their rapid growth and development. This study precisely quantified the muscle fiber number of an important skeletal muscle in poultry for the first time, which provides direct evidence for the physiological basis of rapid development in broilers and offers important data support for further in-depth studies on muscle fiber development.