Field, greenhouse, and laboratory experiments were conducted to investigate resistance to glyphosate in tropical sprangletop biotypes (Lv8 and Lv9) collected in Persian lime from Veracruz, Mexico. Assays to determine the dose required to reduce seedling fresh weight by 50% indicated a resistance factor (RF) of 4.9 and 3.2 for biotypes Lv8 and Lv9, respectively; whereas the LD50 showed a RF of 4.4 and 3.3 for biotypes Lv8 and Lv9, respectively. On the other hand, the RFs using whole plant dose–response assays were lower (RF of 3 for Lv8 and 2.3 for Lv9). The susceptible biotype (LvS) accumulated 5.5 and 11.8 times more shikimate than biotypes Lv8 and Lv9, respectively, at 96 h after treatment (HAT). In field experiments, alternatives to glyphosate-resistant tropical sprangletop management were identified. Indaziflam + glufosinate and paraquat + diuron provided over 80% control of in-field populations of tropical sprangletop at 60 d after treatment (DAT). These results confirmed the first reported case of glyphosate-resistant tropical sprangletop.