Using column alias in WHERE clause of MySQL query produces an error

As Victor pointed out, the problem is with the alias. This can be avoided though, by putting the expression directly into the WHERE x IN y clause:

SELECT `users`.`first_name`,`users`.`last_name`,`users`.`email`,SUBSTRING(`locations`.`raw`,-6,4) AS `guaranteed_postcode`
FROM `users` LEFT OUTER JOIN `locations`
ON `users`.`id` = `locations`.`user_id`
WHERE SUBSTRING(`locations`.`raw`,-6,4) NOT IN #this is where the fake col is being used
(
 SELECT `postcode` FROM `postcodes` WHERE `region` IN
 (
  'australia'
 )
)

However, I guess this is very inefficient, since the subquery has to be executed for every row of the outer query.

You can only use column aliases in GROUP BY, ORDER BY, or HAVING clauses.

Standard SQL doesn't allow you to refer to a column alias in a WHERE clause. This restriction is imposed because when the WHERE code is executed, the column value may not yet be determined.

Copied from MySQL documentation

As pointed in the comments, using HAVING instead may do the work. Make sure to give a read at this question too: WHERE vs HAVING.

Standard SQL (or MySQL) does not permit the use of column aliases in a WHERE clause because

when the WHERE clause is evaluated, the column value may not yet have been determined.

(from MySQL documentation). What you can do is calculate the column value in the WHERE clause, save the value in a variable, and use it in the field list. For example you could do this:

SELECT `users`.`first_name`, `users`.`last_name`, `users`.`email`,
@postcode AS `guaranteed_postcode`
FROM `users` LEFT OUTER JOIN `locations`
ON `users`.`id` = `locations`.`user_id`
WHERE (@postcode := SUBSTRING(`locations`.`raw`,-6,4)) NOT IN
(
 SELECT `postcode` FROM `postcodes` WHERE `region` IN
 (
  'australia'
 )
)

This avoids repeating the expression when it grows complicated, making the code easier to maintain.