<p>Congenital transmission is a key route for <i>Trypanosoma cruzi</i> infection, yet the cellular mechanisms enabling congenital transmission remains poorly understood. This study evaluated the susceptibility of different trophoblast cell line models to <i>T. cruzi</i> infection and compared infectivity patterns among TcI strains compared to Y strain (TcII). The BeWo cell line was used to model cytotrophoblast (CT) and syncytiotrophoblast (ST) cells, while the HTR-8/SVneo cells modeled extravillous trophoblasts (EVTs). Three Colombian TcI strains and one TcII strain were cultured and differentiated into infective trypomastigotes. Infection rates, intracellular parasite replication, trypomastigote release, cell viability, hormone secretion, apoptosis, and ultrastructural characteristics were studied. Significant differences in infection susceptibility among trophoblast types were observed. BeWo CT-like cells were highly permissive to infection, whereas ST-like cells exhibited strong resistance, potentially due to intrinsic defense mechanisms. HTR-8/SVneo showed moderate susceptibility, supporting persistent replication and trypomastigote release. Infection impaired cell viability and disrupted endocrine functions such as hCG secretion, with cell-type-specific effects. Parasite strains also differed in infectivity. Strain Υ demonstrated high infectivity and cytotoxic effects. In contrast, SN3 and GAL61S strains exhibited slower intracellular development and lower release of trypomastigotes. Strain SA showed early infectivity but limited replication. These findings highlight the differential susceptibility of trophoblast subtypes to <i>T. cruzi</i> infection and the variability in strain virulence. Further insight into placental defense mechanisms and parasite-host interactions is essential for understanding congenital transmission and developing targeted preventive strategies.</p>