Acanthaceae has received considerable taxonomic attention at the familial, subfamilial, tribal and subtribal levels. Several different infra-familial classifications have been proposed for the Acanthaceae, but no taxonomic consensus has yet been reached. The main objective of the present study is to throw light on the phenetic relationships and to explore the contribution of morphological and molecular characters in systematics of Acanthaceae. The morphological data viz. macromorphology, stomatography, lamina architecture and ISSR profiles of 30 Egyptian acanthaceous taxa were investigated. The phenetic analysis using NTSYS-PC version 2.02 software based on 55 potentially informative morphological and molecular characters indicated that the used morphological and ISSR criteria is likely to be useful and valuable taxonomic traits. The morphological characters and ISSR aspects of all the studied species produced a phenogram that showed two series; one of them had two subseries, the first one comprised only three taxa while the second divided into two clusters, each contained two groups. The delimitation and the membership of the studied taxa clearly merit additional study using more criteria. The phenetic analysis of both morphological and molecular attributes clarified the segregation of genus Avicennia as a distinct identity away from Acanthaceae. Acanthus mollis & A. montanus are isolated in its own series that comparable to tribe Acantheae of the current taxonomic systems. The studied species of Thunbergia are gathered its own subseries that comparable to tribe Thunbergiae and Ruellia in its own group that comparable to tribe Ruellieae.