African honey bee subspecies Apis mellifera scutellata began to spread in the American continent from southern Brazil in 1956. The process of Africanization involved both maternal and paternal bidirectional gene flow between European and Africanized honey bees. In Argentina, Africanized honey bees dominate in the northern semitropical regions and a hybrid area is defined between (ca. 32°–34°latitude). Although previous analysis has been carried out in Buenos Aires province, no analysis has been made south of this latitude. We used mitochondrial DNA (mtDNA) assays and wing geometric morphometrics to determine the prevalence of Africanized honey bees in managed populations. African mitotype origin and subspecies identity, were determined at three regions from Argentina. In our study, we analyzed 480 samples by wing morphometry and 157 by cytochrome-b assays, collected during 2013–2016. Our results show that North of 35°N latitude, honey bees with African mitotype were quite common (88.64%) finding high similarity with A. m. scutellata. South of that latitude, in temperate regions, we found African mitotype in only 18 of 133 colonies analyzed and a mixture of both European (A. m. ligustica, A. m. cárnica, A. m. mellifera) and African subspecies, most likely derived from North African honey bee (A. m. intermissa). Our results confirmed the existence of Africanized honey bee populations with a gradual cline from north to south, as a result from recent A. m. scutellata-derived bee expansion. Beyond the transition area between the 30°–35°S parallels bees carrying the European mitotype were fairly common.