The Karst area is widely distributed in Yunnan Province, the evaluation of farmland fertility in different counties in karst areas can provide scientific bases for the utilization, improvement and agricultural production layout of soil resources. In this study, 248 soil samples were collected from farmland profiles in Xuanwei, Shilin and Yanshan in the karst areas of central and eastern Yunnan, total nitrogen, available phosphorus, available potassium, organic matter and pH of the samples were determined, and then soil fertilities of farmlands were assessed by the principal component analysis (PCA) method. The results showed that the determined five soil indexes were moderately variable in tillage layers in the three regions. Total nitrogen, available phosphorus, available potassium and organic matter were significantly different between AB layers and A (A1 and A 2) layers, but weren’t significantly different between AB layers and B layers. Three principal components were extracted by PCA which reflected 82.954% of the original information. The first principal component was organic matter and total nitrogen, the second one was available phosphorus, the third one was available potassium, the comprehensive scores of soil fertility was in order of Xuanwei (0.231) > Shilin (-0.289) > Yanshan (-0.335). The above results can provide reference for soil nutrient regulation and developing plateau characteristic agriculture in karst areas.