Handy Plant Efficiency Analyser (Handy PEA) provides a method for the high-throughput screening of photosynthetic germplasm. However, the large number of chlorophyll a fluorescence parameters (CFPs) from PEA and the inconsistency of CFP applications among studies greatly limit the accuracy of photosynthesis analyses. In this study, all 53 CFPs of 186 upland cotton cultivars (strains) were measured at 12:00 and 17:00 h. Thirty-two CFPs were selected according to biological importance, and the CFP relationships were determined. Differences in the response ability of cotton cultivars (strains) to high light intensity stress were demonstrated by the distribution of CFPs. Furthermore, the classification and evaluation of photosynthetic characteristics of cotton cultivars (strains) were carried out by Principal Component Analysis and Cluster Analysis. Finally, ten cotton cultivars (strains) with good photosynthetic performance were selected. This study provides a high-throughput method how to identify cotton germplasm resources with high photosynthetic efficiency.