Lipid droplet-associated proteins (LDAPs) play essential roles in tissue growth and development and in drought stress responses in plants. Cotton is an important fiber and cash crop; however, the LDAP family has not been characterized in cotton. In this study, a total of 14, six, seven, and seven genes were confirmed as LDAP family members inGossypium hirsutum,Gossypium raimondii,Gossypium arboreum, andGossypium stocksii, respectively. Additionally, expansion in the LDAP family occurred with the formation ofGossypium, which is mirrored in the number of LDAPs found in five Malvaceae species (Gossypioides kirkii,Bombax ceiba,Durio zibethinus,Theobroma cacao, andCorchorus capsularis),Arabidopsis thaliana, andCarica papaya. The phylogenetic tree showed that theLDAPgenes in cotton can be divided into three groups (I, II, and III). The analysis of gene structure and conserved domains showed thatLDAPsderived from group I (LDAP1/2/3) are highly conserved during evolution, while members from groups II and III had large variations in both domains and gene structures. The gene expression pattern analysis ofLDAPgenes showed that they are expressed not only in the reproductive organs (ovule) but also in vegetative organs (root, stem, and leaves). The expression level of two genes in group III,GhLDAP6_At/Dt, were significantly higher in fiber development than in other tissues, indicating that it may be an important regulator of cotton fiber development. In group III,GhLDAP2_At/Dt, especiallyGhLDAP2_Dtwas strongly induced by various abiotic stresses. Decreasing the expression ofGhLDAP2_Dtin cottonviavirus-induced gene silencing increased the drought sensitivity, and the over-expression ofGhLDAP2_Dtled to increased tolerance to mannitol-simulated osmotic stress at the germination stage. Thus, we conclude thatGhLDAP2_Dtplays a positive role in drought tolerance.