Abiotic stress induced plasma membrane protein 3 (PMP3) genes occur as multigene families in plants, coding for hydrophobic proteins. Group I PMP3s code for shorter ORFs while Group II PMP3s code for proteins with C-terminal extensions. Allotetraploid Nicotiana tabacum (SSTT; 2n = 48) derives its parentage from extant ancestors related to Nicotiana sylvestris (SS) and Nicotiana tomentosiformis (TT). Polyploidization triggers complex genetic and epigenetic changes, often leading to homoeolog-specific retention or loss of function, sub-functionalization or neo-functionalization. Genomic sequences of Nt(t)PMP3-1/Nt(t)PMP3-2 cloned from N. tabacum show near identity with N. tomentosiformis NtoPMP3-1/NtoPMP3-2 genomic sequences respectively (distinct from N. sylvestris NsPMP3-1/NsPMP3-2 genomic regions). RT-PCR with exon 1,2 primer pairs amplified only single fragments for Nt(t)PMP3-1 and Nt(t)PMP3-2. In contrast, for Nt(t)PMP3-2, three variants were detected using exon 2,3 primers by RT-PCR. Cloning revealed (i) a transcript coding for a Group I PMP3 [Nt(t)PMP3-2CS], (ii) a transcript with complete retention of the second intron [Nt(t)PMP3-2IR] and (iii) a transcript with an alternative (exon 2) 5′ splice site [Nt(t)PMP3-2AS], coding for a longer protein, similar to ORFs of Group II PMP3 genes. All three Nt(t)PMP3-2 variants have conserved counterparts in the N. tomentosiformis transcriptome, suggesting the transcriptional machinery governing alternative splicing of Nt(t)PMP3-2 in N. tabacum has conserved origins, derived from a N. tomenosiformis lineage. The above data shows alternative splicing of PMP3 genes contributes to transcript and ORF diversity in plants. All three Nt(t)PMP3-2 splice variants show increased root-specific expression. Implications of Nt(t)PMP3-2 alternative splicing on transcript stability and ORF features are discussed.