ndhF-rpl32 is a noncoding region of chloroplast genome and because of its high rate of nucleotide substitution it has been recommended to investigate the relationships at lower taxonomic level i.e., between species, genus and tribe. In the current study this region has been explore to infer the relationships at subfamilial level in Solanaceae. Eighteen species of Solanaceae and one Species of Convolvulaceae were included in the study. The phylogenetic analysis using sequence data of intergenic spacer ndhF-rpl32 was performed followed by DNA extraction and DNA sequencing. The results indicated that Solanaceae was a monophyletic family and consist of three subfamilies; Solanoideae, Cesteroideae and Nicotinoideae. Within Solanoideae five tribes were observed namely; Solaneae, Capciceae, Lycieae, Physaleae, Datureae. This spacer region resolve the association between species of different genera with strong support. Itresolve the association between genera of tribe Physaleae (Withania and Physalis). It also provide greater resolution among lineages of subfamilies; Solanoideae, Cesteroideae and Nicotinoideae. However, it poorly resolve the association between tribe of subfamily Solanoideae. Overall results exhibited that ndhF-rpl32 was able to provide great resolution at species, genus and subfamily level but not at tribe level. The results indicated that the spacer region could be utilized to explore the association at subfamilial level in Solanaceae. The increased number of sample size may also provide clearer picture of the association.