Prevent users from uploading the same file twice using EnableDuplicateCheck="true". Use DuplicateHashAlgorithm to specify the hashing method for content-based detection.

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<au:AjaxFileUpload ID="Uploader1" runat="server"
 AllowMultiple="true"
 EnableDuplicateCheck="true"
 DuplicateHashAlgorithm="sha256"
 ButtonText="Select Files"
 AutoUpload="true"
 ShowProgress="true" />

Try selecting the same file twice to see it detected as a duplicate.