To explore the resistance function of chitinase to gray mold disease in Lilium spp., we cloned a chitinase gene LoChi2 (GenBank accession number: MW310626) by reverse transcription PCR from the highly resistant cultivar L. oriental hybrid ‘Sorbonne’ after Botrytis elliptica infection for 12 h. Structural and functional characteristics of the protein encoded by LoChi2 gene were predicted through bioinformatic methods, and the expression patterns of this gene in response to B. elliptica infection and JA/SA treatment were analyzed by qRTPCR. The results shows: (1) the open reading frame of LoChi2 is 915 bp long and encodes 304 amino acids with a predicted molecular weight of 32.52 kD and a calculated isoelectric point of 4.16. (2) Protein structural and phylogenetic analyses revealed that LoChi2 is a member of Class Ⅲ from glycoside hydrolase 18 family. As a secretory protein with hydrophobicity, LoChi2 is located outside the cell. It includes a conserved GH18 narbonin catalytic domain, a transmembrane domain, as well as signal peptide, glycosylation, and phosphorylation sites. Multiple sequence alignment of the deduced amino acid sequence of LoChi2 displayed high similarity with the sequences of several Chi2 genes from L. longiflorum, Ananas comosus, and Prunus mume. (3) qRTPCR analysis showed that the expression levels of LoChi2 were positively correlated with gray mold disease resistance among different cultivars. More transcripts of LoChi2 accumulated in the highly resistant cultivar ‘Sorbonne’ than in the moderately susceptible L. × formolongi ‘Raizan 3’ and highly susceptible L. asiatic hybrid ‘Tresor’ after infected with B. elliptica; the expression of LoChi2 was induced by exogenous salicylic acid and jasmonic acid treatments. Overall, our findings indicate that LoChi2 is a critical gene involved in lily defense against gray mold disease, and this gene may play an important role in the JA and SA mediated defense signaling pathways. We thus speculate that it will become a new candidate gene with broad application prospects in the transgenic breeding of lily resistance to this disease.