Daylily rust—caused by Puccinia hemerocallidis—was confined to Eastern Asia until the disease was reported in Oceania, Africa, the Americas and Portugal in the 21st century. Although information on rust resistance of American cultivars is available, little is known about the resistance of European bred cultivars, threating the ornamental sector if the fungus spreads to other European countries. Aiming to provide tools to address this, we analyzed the Portuguese pathogens and characterized rust resistance in a selection of cultivars, while optimizing disease rating scales. Morphologic, genetic and cytogenomic characterization of four isolates reveals narrow diversity and raises the question whether the pathogen may have originated in North- or Central America. Daily records of multiple symptomatologic parameters enabled a detailed disease progress analysis, discriminating cultivars according to their resistance levels and revealing susceptibility as the most common state. Among the tested cultivars, 12 out of 17 began to show symptoms between 6–8 dai and were classified as susceptible. Cultivars ‘Stella d’Oro’, ‘Bitsy’ and ‘Cherry Tiger’ behaved as moderately resistant although the occurrence of late sporulation on leaves suggests incomplete resistance and challenges common rating scales. The identification of resistance sources in European breeding lines is crucial for the sustainable future of daylilies.