Casuarina equisetifolia has the widest distribution of all Casuarina species and is a nitrogen-fixing tree of considerable social, economic and environmental importance. Trichosporium vesiculosum, a causal agent of blister bark disease, is a serious pathogen of C. equisetifolia. In this study, a cDNA clone encoding class I chitinase (CeChi1) belonging to PR-3 family was cloned and characterized from the needle tissues of C. equisetifolia challenged with the toxic exudate of the fungal pathogen T. vesiculosum. The CeChi1 open reading frame comprised 966 nucleotides that encoded 321 amino acid residues with the molecular mass of mature protein being approximately 34 kDa. Analysis of the predicted amino acid sequence revealed the similarity of CeChi1 protein to class I chitinase from other plant species containing a hydrophobic signal peptide domain and hinge domain. The sequence also harboured a cysteine-rich chitin-binding domain and lysozyme-like domain. A hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants was also detected. The genomic sequence of CeChi1 indicated that the coding region contained three exons and two introns. In silico analysis of the untranslated regions revealed the presence of several cis-acting regulatory elements associated with hormonal regulation and stress responses. Quantitative real-time PCR analyses at different time points showed upregulation of the transcript during pathogen elicitation and salicylic acid signalling. However, no significant expression of CeChi1 was observed during other abiotic stress condition including wounding, water deficit, salt and heat stress revealing the specific expression of the gene during pathogenesis. This is the first report on isolation of a gene from C. equisetifolia, and the detailed functional analyses of CeChi1 will help in understanding its specific role in defence against pathogens in this tropical tree species.