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Contents The aim of this study was to develop a non‐invasive procedure to stimulate ejaculation in median and small lizards for semen collection. After semen collection, we applied a battery of tests to assess the motility (wave and progressive), viability (eosin–nigrosin stain), morphology (normal/abnormal), sperm concentration and ejaculate number, seminal volume and colour. We obtained this fluid from all males of the four species of sceloporine lizards (n = 30) and one species of Mexican horned lizards (n = 7). We found that semen from all males had a liquid‐like consistency and a milky‐white appearance. Ejaculate volume and sperm concentration varied among the males and the species studied. We also observed that although the mobility, viability and normal sperm morphology showed a wide variability, their average value in each species was high. We conclude that “genital papilla pressure” was an efficient and non‐invasive semen obtaining method for small‐ and medium‐sized lizards that does not imply damage to donors and could be used in other lizard species.