Enterocytozoon hepatopenaei (EHP) is a pervasive microsporidian causing chronic sublethal effects on growth, feed utilization, and survival in Penaeus vannamei, posing significant challenges to shrimp farmers. This study evaluated three commercial genetic lines under a controlled EHP challenge using feces-top-coated feed to mimic natural infection while ensuring controlled exposure. All three lines demonstrated similar performance in negative control tanks. Following challenge, Line C consistently outperformed the other lines in survival, growth, feed conversion ratio (FCR), and delayed pathogen proliferation. After 28 days post-infection, Line C shrimp achieved 90.00 ± 1.99% survival, 5.80 ± 0.34 g growth, and an FCR of 1.26, whereas Lines A and B showed statistically significant lower survival (45.19–53.65%) and growth. Quantitative PCR analyses indicated that early pathogen replication was strongly suppressed in Line A. Additional research could explore possible host mechanisms in tolerant lines that delay infection and preserve hepatopancreatic function. This delayed disease progression allowed biomass accumulation prior to full EHP impact. Under commercial conditions, levels of tolerance demonstrated in the current study would reduce production losses, providing economic benefit to farmers. These results reveal substantial genetic variation in tolerance to chronic EHP infection and highlight the potential value of genetic selection for improving tolerance to EHP in shrimp aquaculture.