Yes, it is possible to add a separator between items in a Python add-in wizard. The config.xml file for the add-in allows for the property, separator="true", to be added into an xml tag to add a separator before an item in the menu. However, there are certain cases where the separator property ends up in a different tag than where it belongs. For example, the separator tag may exist in the following example of a menu tag:
<Menu caption="Submenu1" category="Test Addin" id="AddIns_addin.submenu1" isRootMenu="false" isShortcutMenu="false" separator="true">
Instead, the separator property must be set in the following tag. The example shown is used to set the separator value to true for a Menu item:
<Menu refID="AddIns_addin.submenu1" separator="true"/>
The separator tag can also be added into a Button item as shown in the following example:
<Button refID="RJSS.button2" separator="true"/>
For ArcGIS for Desktop versions prior to 10.3, separators between buttons are not supported in Python add-ins.
In addition, the separator property must be included in every sub-menu for the separation to be visible. If the separator property is only set in the first sub-menu, the separator does not appear since no items precede it in the toolbar. The separator property is used to add a separation before the item in the tag.