Multiple, highly similar members of the lipid transfer protein (LTP) family have been identified in Nicotiana glauca L. Here we describe four new members of the NgLTP gene family and further characterise one member. Three genes were isolated from a guard cell cDNA library and one (NgLTP2) was isolated from a genomic library. These four NgLTPs, as well as one described previously, NgLTP1, share >83% amino acid similarity, but the deduced protein sequence of NgLTP2 lacks the last five residues compared with other LTPs. Since the DNA sequences of the five genes are nearly identical, techniques based on nucleic acid hybridisation or PCR amplification were not sufficient to resolve the expression of the individual genes with confidence. Therefore, we characterised the expression pattern of NgLTP2, the only NgLTP gene that was not found in the guard cell cDNA library, using an NgLTP2 promoter-GUS reporter assay. GUS activity driven by the NgLTP2 promoter was assayed in three species of transgenic plants as an indicator of the endogenous pattern of expression of this gene. GUS was strongly induced upon wounding, whereas NgLTP1 was induced by drought stress. Sequence analysis of the NgLTP2 promoter revealed cis-acting motifs associated with induction by wounding. Differential expression of the NgLTP gene family, revealed by the different expression patterns of NgLTP1 and NgLTP2, is further evidence that these genes have multiple functions in N. glauca.