Recent researches focused on breeding lycopene–rich watermelon varieties are active since lycopene content in watermelon is 60% higher than in tomato. We developed a new microplate method using a UV spectrophotometer to select genetic resources of watermelon containing high lycopene content. The microplate method showed more accurate analysis (about 97% positive correlation) than HPTLC, suggesting the microplate method is a reliable analytical technique for the rapid and simple selection of watermelon resources. Lycopene contents of 96 watermelon accessions using the new microplate method was a wide variation of 3.2-98.3 μg.g-1FW to assess, allowing selection of 15 high-lycopene watermelon accessions. Lycopene contents of the 15 watermelon accessions was 4.4~9.6 fold higher than lycopene content of a commercial cultivar ‘Speedplus’. These results suggest that the watermelon accessions selected using a novel microplate method can be used for breeding of lycopene-rich cultivars.