The phylogenetic relationships within Apocynaceae were investigated in the present study. In addition trnL-F intron-spacer region and atpB promoter, a part of PHYA exon, a low-copy nuclear gene were sequenced from Apocynaceae. Different taxa of the family were collected from Pakistan and different regions of the world, representing major groups of the family. Separate phylogenetic trees were constructed using trnL-F and PHYA sequences and then combined datasets were used for simultaneous analysis. In the separate trnL-F analyses (comprised of 178 taxa with updated nomenclature) both parsimony and Bayesian, yield a number of stable clades, but placement of tribes (Vinceae, Tabernaemontaneae, Hunterieae and Melodineae) in Rauvolfioideae is uncertain and lack high level of support. A clade comprising Ceropegieae and Marsdenieae receives good support confirming the monophyly of both tribes. The grouping of taxa in Asclepiadeae is not satisfactory to define a subtribal classification. Malagasy Cynanchum group forms a separate clade in both analyses while the monophyly of New World Cynanchineae is not supported here. In the combined phylogenetic analyses, 112 taxa were included representing most major caldes in Apocynaceae. The study confirms that Periplocoideae are nested within Apocynoideae. The APSA clade (Apocynoideae, Periplocoideae, Secamonoideae and Asclepiadoideae) is strongly supported here, but the crown clade of Apocynaceae (comprised of subfamilies Asclepiadoideae, Secamonoideae, Periplocoideae and Echiteae, Mesechiteae, Odontadenieae and Apocyneae of Apocynoideae) has only moderate support. The present study places Periplocoideae as part of the sister group to the rest of the crown clade and the tribe Baisseeae emerges as sister group of Secamonoideae-Asclepiadoideae clade. Old World Cynanchinae form a well-supported clade with the New World MOG (Metastelmatinae, Oxypetalinae and Gonolobinae) tribes rather than with the largely Old World Asclepiadinae and Tylophorinae, as suggested by earlier studies. By addition of atpB promoter sequences of Rauvolfioideae’s taxa in combined dataset (trnL-F and PHYA), the inter-generic resolution was not improved in the subfamily. In the present study, resolution among most groups (such as inter-tribal relationships of Asclepiadoideae) is improved in combined analyses as compared to previous phylogenies, based on only plastid regions. However, there is a need to sequence vmore nuclear loci like PHYA from greater number of taxa to further improve relationships in the family.