The first reference that I could find there are lots later that says that Tux is definitely male comes in this comment from Linus most of the time hes been saying it  but here for the first time that I can find he calls the penguin a he   Ooh what a cuddly penguin I bet he is just stuffed with herring and small children will jump up and down and scream mommy mommy can I have one too  That dates back to an extremely famous email entitled Re Linux Logo prototype from Linus Torvalds torvaldscshelsinkifi  dated Thu  May    EET DST  from the Linux kernel mailing list  which you can find archived here  httplkmlindianaeduhypermaillinuxkernelhtml  you can find many MANY mentions of the penguin with male gender language laterbut this is probably the first