Agalinis (Orobanchaceae, tribe Pedicularideae) consists of approximately 70 hemiparasitic species distributed among North, Central, and South America. Previous phylogenetic studies did not include tropical species nor comprehensive sampling for closely allied genera, limiting our systematic understanding of these lineages. We generated an updated phylogenetic hypothesis from 179 individuals from 51 species of Agalinis and five genera once considered congeneric with Agalinis (Anisantherina, Brachystigma, Dasistoma, Aureolaria, and Esterhazya), particularly focusing on sampling underrepresented Central and South American taxa. Phylogenetic analyses were based on six cpDNA regions (rbcL, matK, trnT(UGU)-trnF(GAA), rps2, rpoB, and psbA-trnH) and four nuclear regions (ITS, PPR-AT1G09680, PPR-AT3G09060, and PPR-AT5G39980) and were conducted using maximum likelihood and Bayesian inference methods. Additionally, our data were added to previously published Orobanchaceae-wide datasets to reveal placement of these lineages in a comprehensive phylogenetic context using maximum likelihood. Our results reveal a strongly supported South American Agalinis clade that is sister to taxa from southwestern North America and Mexico and includes distinct Brazilian and Andean clades. Esterhazya, a hummingbird-pollinated genus in Brazil, is nested within the Brazilian Agalinis clade with strong support. We confirm well-supported incongruences between chloroplast and nuclear partitions, particularly concerning placement of early-diverging lineages within Agalinis. The monotypic genus Anisantherina, once included in Agalinis, is resolved within Orobanchaceae tribe Buchnereae, a placement that is further corroborated by anther morphology. Orobanchaceae consist of numerous species from tropical regions that have not been included in phylogenetic studies, and our study underscores the importance of more comprehensive sampling for the placement of these lineages to clarify taxonomy, biogeography, and character evolution.