The present study aimed at characterizing the composition and expression level of Collagen type III (COL3) in chicken Pectoralis major muscles (PMs) affected by wooden breast (WB), to ultimately deepen the knowledge on its potential implication in the onset of this myopathy by specifically addressing the hypothesis of a different COL3 biosynthesis and composition in WB breasts. To this purpose, 10 PMs (5/group) were selected 3 hours post-mortem at a commercial broiler abattoir, grouped according to their phenotype as unaffected (N) or severe WB, and collected for subsequent analysis. From each sample, an aliquot was subjected to COL3 extraction and amino acid composition analysis. An additional subsample (flash-frozen in liquid nitrogen; stored at -80°C) was used to perform gene expression analysis. More in detail, COL3 amino acid composition was assessed by HPLC method, and the normalized gene expression level of its coding gene (COL3A1) was quantified by means of qRT-PCR. Furthermore, normalized expression of Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) and Lysyl oxidase (LOX) mRNA were investigated in view of their post-translational role in collagen synthesis and stabilization. For gene expression analyses, GAPDH has been used as normalizing gene. After testing the normality of data distribution (Shapiro-Wilk test), differences between groups were assessed by using the non-parametric Mann-Whitney U test. Statistical analyses were performed in R (v.4.3.2) and all differences were considered significant at a level of P≤0.05. Results from the COL3 amino acid composition evidenced that WB-affected PMs had a higher (P<0.05) content (g/100g of COL3) of glycine (14.6 vs 10.5), proline (7.2 vs 4.9), alanine (5.6 vs 4.1), hydroxyproline (7.9 vs 5.7) and hydroxylysine (1.2 vs 0.7) when compared with N. As for the gene expression results, COL3A1 normalized expression was significantly higher (P≤0.05) in WB compared to N samples. Analogously, both LOX and P4HA1 normalized levels of expression were significantly higher (P≤0.05) in WB than N. Overall, results achieved in this study corroborated previous evidence that the occurrence of WB is strongly related to alterations in connective tissue components of PMs. Besides, in view of its association with muscle regeneration, the higher expression level of COL3 in WB muscles might be associated with the pathological events occurring in abnormal muscles. However, extensive alterations in COL3 composition together with profound changes in gene expression levels of players involved in its post-translational modifications might suggest its involvement in the WB underlying mechanisms. Research supported by NextGenerationEU, National Grant PRIN2022 (Prot. n. 2022EPWEPW).