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To assess the genetic diversity among lotus (Nelumbo) accessions and evaluate the correlation between genetic variation and morphological classification, we sampled 138 accessions: two of N. lutea, 112 of N. nucifera, 17 of hybrids between N. nucifera and N. lutea, and seven Japanese cultivars. The 11 selected combinations of amplified fragment length polymorphism (AFLP) primers produced 138 polymorphic loci, and the percentage of polymorphism was 28.7%. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram clustered all the accessions into two groups: Group I comprised N. lutea and its hybrids with N. nucifera; Group II included N. nucifera and its hybrids with N. lutea and Japanese cultivars. Population structure analysis identified four main clusters: N. lutea clustered mainly in C1, whereas N. nucifera clustered in C2, C3, and C4, which was consistent with the UPGMA and principal coordinate analysis results. The Japanese cultivars were related more closely to N. nucifera (genetic similarity coefficient = 0.74) than to N. lutea (0.46); hence, the Japanese cultivars can be classified as N. nucifera. Moreover, rhizome lotuses formed a separate subclade, whereas seed lotuses were interspersed among flower lotuses, which demonstrated that rhizome lotuses were distinct from flower and seed lotuses. Plant size, flower color, and other morphological criteria used commonly to classify lotuses were correlated with genetic variation to a certain extent but not sufficiently for accurate classification. It appears that it is necessary to use both DNA markers and morphological characteristics to classify lotus species and cultivars.