Here we report the results of fine resolution Y chromosomal analyses (Y-SNP and Y-STR) of 267 Bantu-speaking males from three populations located in the southeast region of Africa. In an effort to determine the relative Y chromosomal affinities of these three genotyped populations, the findings are interpreted in the context of 74 geographically and ethnically targeted African reference populations representing four major ethno-linguistic groups (Afro-Asiatic, Niger Kordofanin, Khoisan and Pygmoid). In this investigation, we detected a general similarity in the Y chromosome lineages among the geographically dispersed Bantu-speaking populations suggesting a shared heritage and the shallow time depth of the Bantu Expansion. Also, micro-variations in the Bantu Y chromosomal composition across the continent highlight location-specific gene flow patterns with non-Bantu-speaking populations (Khoisan, Pygmy, Afro-Asiatic). Our Y chromosomal results also indicate that the three Bantu-speaking Southeast populations genotyped exhibit unique gene flow patterns involving Eurasian populations but fail to reveal a prevailing genetic affinity to East or Central African Bantu-speaking groups. In addition, the Y-SNP data underscores a longitudinal partitioning in sub-Sahara Africa of two R1b1 subgroups, R1b1-P25* (west) and R1b1a2-M269 (east). No evidence was observed linking the B2a haplogroup detected in the genotyped Southeast African Bantu-speaking populations to gene flow from contemporary Khoisan groups.