Crop adaptation to environmental changes showed a short-term effect characterized by physiological and biochemical changes, and a long-term effect characterized by changes in dry matter accumulation. The effects of suboptimal low temperature (15 °C/10 °C, day/night, 5 days) on physiological characters, growth and development were investigated in 16 genotypes of melon seedlings. For short-term effect, suboptimal low temperature mainly induced free proline (up to 179.5%) and soluble sugar accumulated (up to 181.4%), impaired plasma membrane, and inhibited chlorophyll biosynthesis and PSII activity. For long-term effect, it mainly inhibited plant height and the first female flower opening (FFFO), but promoted stem diameter growth. Comprehensive analysis showed that nine indicators were suitable for assessing suboptimal low temperature effect on melon seedlings, including chlorophyll content (total, chl.a and chl.b), plant height, internode length, stem diameter, free proline, soluble sugar, and FFFO. Furthermore, sixteen genotypes were divided into four groups based on the nine parameters. Cluster I was characterized by high chlorophyll content and plant height (two genotypes); Cluster II was mainly characterized by intermediate value in 7 - 8 indexes (five genotypes); Cluster III was characterized by most indexes were about one third of the maximum value (seven genotypes); Cluster IV was characterized by lower value in all indexes (two genotypes). Our research can provide detailed data to clarify the response characteristics of different melon populations to suboptimal low temperature, and provide reference for cold acclimation of melon and other cucurbits in practice.