Vaccinium arboreum (VA) is a wild blueberry species that exhibits wider soil pH tolerance and greater ability for iron and nitrate uptake than cultivated Vaccinium species, including southern highbush blueberry (SHB, V. corymbosum interspecific hybrids). The ability of VA and SHB to respond to iron deficiency by rhizosphere acidification was investigated. Rooted cuttings of the VA genotype FL09-502 and SHB ‘Emerald’ were transplanted to a hydroponic system filled with complete nutrient solution. After 14 days of acclimation at 45 µm iron, plants were transferred to unbuffered nutrient solutions containing 90 or 10 µm iron. ‘Emerald’ and FL09-502 plants grown in 10 µm iron exhibited less iron uptake and lower chlorophyll, total iron, and active iron contents than plants grown in 90 µm iron. Generally, there were no species-level differences in iron or nitrate uptake. Neither FL09-502 nor ‘Emerald’ acidified the rhizosphere in either the nutrient solution or in a gel-based assay, regardless of external iron concentration. A screen of 18 additional genotypes of VA and SHB confirmed that this response is absent in these taxa. Thus, rhizosphere acidification is not part of the iron deficiency response of SHB and VA. In addition, the ability to acidify the soil is not likely to be responsible for the wider soil pH tolerance of VA.