Lactation traits are economically important for dairy cows. Southern China has a high-temperature and high-humidity climate, and environmental and genetic interactions greatly impact dairy cattle performance. The aim of this study was to identify novel single-nucleotide polymorphism sites and novel candidate genes associated with lactation traits in Chinese Holstein cows under high-temperature and humidity conditions in southern China. A genome-wide association study was performed for the lactation traits of 392 Chinese Holstein cows, using GGP Bovine 100 K SNP gene chips. Some 23 single nucleotide polymorphic loci significantly associated with lactation traits were screened. Among them, 16 were associated with milk fat rate, 7 with milk protein rate, and 3 with heat stress. A quantitative trait locus that significantly affects milk fat percentage in Chinese Holstein cows was identified within a window of approximately 0.5 Mb in the region of 0.4-0.9 Mb on Bos taurus autosome 14. According to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, ten genes (DGAT1, IDH2, CYP11B1, GFUS, CYC1, GPT, PYCR3, OPLAH, ALDH1A3, and NAPRT) associated with lactation fat percentage, milk yield, antioxidant activity, stress resistance, and inflammation and immune response were identified as key candidates for lactation traits. The results of this study will help in the development of an effective selection and breeding program for Chinese Holstein cows in high-temperature and humidity regions.