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The genus <i>Calonectria</i> includes pathogens of various agricultural, horticultural, and forestry crops. Species of <i>Calonectria</i> are commonly collected from soils, fruits, leaves, stems, and roots. Some species of <i>Calonectria</i> isolated from soils are considered as important plant pathogens. Understanding the species diversity and distribution characteristics of <i>Calonectria</i> species in different soil layers will help us to clarify their long-term potential harm to plants and their patterns of dissemination. To our knowledge, no systematic research has been conducted concerning the species diversity and distribution characteristics of <i>Calonectria</i> in different soil layers. In this study, 1000 soil samples were collected from five soil layers (0–20, 20–40, 40–60, 60–80, and 80–100 cm) at 100 sampling points in one 15-year-old <i>Eucalyptus urophylla</i> hybrid plantation in southern China. A total of 1037 isolates of <i>Calonectria</i> present in all five soil layers were obtained from 93 of 100 sampling points. The 1037 isolates were identified based on DNA sequence comparisons of the translation elongation factor 1-alpha (<i>tef1</i>), β-tubulin (<i>tub2</i>), calmodulin (<i>cmdA</i>), and histone H3 (<i>his3</i>) gene regions, as well as the combination of morphological characteristics. These isolates were identified as <i>C. hongkongensis</i> (665 isolates; 64.1%), <i>C. aconidialis</i> (250 isolates; 24.1%), <i>C. kyotensis</i> (58 isolates; 5.6%), <i>C. ilicicola</i> (47 isolates; 4.5%), <i>C. chinensis</i> (2 isolates; 0.2%), and <i>C. orientalis</i> (15 isolates; 1.5%). With the exception of <i>C. orientalis</i>, which resides in the <i>C. brassicae</i> species complex, the other five species belonged to the <i>C. kyotensis</i> species complex. The results showed that the number of sampling points that yielded <i>Calonectria</i> and the number (and percentage) of <i>Calonectria</i> isolates obtained decreased with increasing depth of the soil. More than 84% of the isolates were obtained from the 0–20 and 20–40 cm soil layers. The deeper soil layers had comparatively lower numbers but still harbored a considerable number of <i>Calonectria</i>. The diversity of five species in the <i>C. kyotensis</i> species complex decreased with increasing soil depth. The genotypes of isolates in each <i>Calonectria</i> species were determined by <i>tef1</i> and <i>tub2</i> gene sequences. For each species in the <i>C. kyotensis</i> species complex, in most cases, the number of genotypes decreased with increasing soil depth. The 0–20 cm soil layer contained all of the genotypes of each species. To our knowledge, this study presents the first report of <i>C. orientalis</i> isolated in China. This species was isolated from the 40–60 and 60–80 cm soil layers at only one sampling point, and only one genotype was present. This study has enhanced our understanding of the species diversity and distribution characteristics of <i>Calonectria</i> in different soil layers.