Huperzine A (Hup A), the alkaloid produced by the Chinese medicinal plant Huperzia serrata, has been documented to be a promising agent for the treatment of Alzheimer's disease due to its potent acetylcholinesterase inhibitory (AChEI) activity. The search for anticholinesterase natural products, as well as for alternative sources of Hup A in Mexican lycopods, prompted us to investigate these plants. The action of methanolic and alkaloidal extracts of three Huperzia species (H. cuernavacensis, H. dichotoma, and H. linifolia) was evaluated using an in vitro anticholinesterase activity assay. Also, chromatographic and spectroscopic analyses were employed to detect the presence of Hup A. Methanolic and alkaloidal extracts of H. cuernavacensis showed IC50 =5.32±0.8μg/mL and 0.74±0.05μg/mL; H. dichotoma displayed AChEI with IC50 values =14.11±2.1μg/mL and 0.64±0.09μg/mL; and H. linifolia presented IC50 =158.37±8.7μg/mL and 4.2±1.24μg/mL, respectively, compared to the control Hup A (IC50= 0.16±0.03μg/mL). Hup A was identified in the extracts of H. dichotoma, but it was not detected in the extracts of H. cuernavacensis and H. linifolia by 1H NMR techniques. This study reveals H. dichotoma as a new source of Hup A, and presents H. linifolia and H. cuernavacensis as potential candidates to obtain other anticholinesterase compounds useful in the Alzheimer's disease treatment.