Lily (Lilium spp.) is a popular ornamental plant. Traditional genetic transformation methods have low efficiency in lily, thus development of a high-efficiency genetic transformation system is important. In this study, a novel transient transformation method involving pollen magnetofection was established and optimized pollen viability, and exogenous gene expression in magnetofected pollen and that of different germplasm were assessed. The highest germination percentage of Lilium regale pollen was 85.73% in medium containing 100 g/L sucrose, 61.5 mg/L H₃BO₃, and 91.5 mg/L CaCl₂. A 1:4 ratio of nanomagnetic beads to DNA plasmid and transformation time of 0.5 h realized the highest transformation efficiency (88.32%). The GFP activity in transformed pollen averaged 69.66%, while that of the control pollen was 0.00%. In contrast to the control, transgenic seedlings obtained by pollination with magnetofected pollen showed strong positive GUS activity with 56.34% transformation efficiency. Among the lily germplasm tested, ‘Sweet Surrender’ and L. leucanthum had the highest transformation efficiency (85.80% and 54.47%), whereas L. davidii var. willmottiae was not successfully transformed. Transformation efficiency was positively correlated with pollen equatorial diameter and negatively correlated with polar axis/equatorial diameter ratio. The results suggest that pollen magnetofection-mediated transformation can be applied in Lilium but might have species or cultivar specificity.