The diseases associated with fowl adenovirus (FAdV) infection, such as inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HPS), and gizzard erosion (GE), were first reported in Pakistan in 1987, and subsequent outbreaks have been reported worldwide, especially in China, where severe outbreaks of HPS with high mortality from 30 to 100% were recently reported and resulted in significant economic losses to the poultry industry. The diagnosis methods of FAdVs were mostly limited to the nucleotide sequence of hexon by PCR and DNA sequencing. The aim of this study was to generate B cell epitope maps of the species- and serotype-specific hexon L1 using monoclonal antibodies (mAbs) and bioinformatics tools for the development of novel diagnostic methods. In this study, the hexon L1 (230 amino acids) was expressed and used to generate 10 mAb-expressing hybridoma cell lines against the relative protein peptide. Subsequently, we defined the linear peptide epitopes recognized by these mAbs using a series of partially overlapping peptides derived from the FAdV-C hexon protein amino acid sequence to map mAbs reactivity. Finally, a common B cell epitope ((31)PLAPKESMFN(40)) for all species FAdVs and two FAdV-C-specific epitopes ((79)KISGVFPNP(87) and (181)DYDDYNIGTT(190)) were identified. These mAbs and their defined epitopes may support the development of the universal or species-specific differential diagnostic methods of FAdVs.