Knowledge of molecular mechanism(s) implicated in smokeless tobacco (ST) associated oral carcinogenesis is meager. In an attempt to identify genes that are modulated by ST, we recently reported establishment of an oral epithelial cell culture, AMOL III from oral hyperplasia with hyperkeratosis of a khaini consumer. Herein we aimed to identify novel molecular targets of ST (khaini) in AMOL III cells using differential display. Fourteen novel differentially expressed genes (12 upregulated and 2 downregulated) were identified. These differentially expressed cDNAs were amplified, cloned, sequenced and confirmed by reverse northern blotting. Mainly these genes are components of transcriptional machinery, cell-cell adhesion, signaling, growth and transformation processes. The important novel molecular targets identified included activated leucocyte cell adhesion molecule (ALCAM), CDP-diacylglycerol-inositol 3-phosphatidyl transferase (phosphatidylinositol synthase), CDIPT, an important enzyme in phosphatidyl inositol biosynthesis, ribosomal protein (RPS23), KIAA0121 and growth and transformation factor, E2IG5. Semi-quantitative RT-PCR analysis of these five genes confirmed over-expression of these genes in oral pre-malignant lesions (OPLs) and oral squamous cell carcinomas (OSCCs) of ST consumers underscoring their biological relevance in ST-associated oral tumorigenesis. In depth studies are warranted to determine the functional significance of ALCAM and CDIPT in oral carcinogenesis.