Human immunodeficiency virus type 1 (HIV-1) can be acquired through oropharyngeal tissues in breastfeeding infants. Efforts to better understand the determinants of breast milk transmission are hampered by the lack of a relevant oral human mucosa model and well-defined breast milk-derived viruses. This study used human ex vivo palatine tonsil tissues and peripheral blood mononuclear cells (PBMCs) to characterize the genetic, biological, and replicative properties of HIV-1 variants obtained from a perinatal transmission pair. Unique viral populations from maternal breast milk and infant blood were identified by gp120 V1-V2- and V3-specific heteroduplex tracking assays (HTAs). Full-length infectious recombinant viruses, containing a common HIV-1 NL4-3 genetic background, were generated with V1-V3 gp120 fragments from maternal and infant isolates representing the major viral populations identified in the HTAs. The resulting recombinant viruses used the CCR5 coreceptor, were nonsyncytium forming, and demonstrated replication properties similar to those of parental and control viruses in PBMCs and tonsillar explants. These findings indicate that viruses from breast milk cells and infant blood can infect PBMCs and tonsil tissues. The maternal and infant HIV-1 viruses detailed here will provide useful tools for defining the viral and host factors that contribute to HIV breastfeeding transmission.