Yes, it matters.
null is a value - The variable has been defined but not set to anything.
defined means exactly what it says - The variable has been defined (to any value).
Try this experiment... Without setting myVar, put this in your template:
{{ (myVar is defined) }}
Since that equates to false, nothing will appear in your template. Now change it to this:
{{ (myVar is null) }}
That will actually throw an error, since myVar was never defined.
To further elaborate, null is a value (as previously mentioned), while defined is a test.
http://twig.sensiolabs.org/doc/tests/defined.html
In PHP terms, it's like the difference between is_null() vs. checking whether the value exists in get_defined_vars() (see this Stack Overflow thread for a little more clarity)