Sure, just set the (appropriately named) -background option to whatever you want.

#!/usr/bin/perl
use warnings;
use strict;
use Tk;

my $top = MainWindow->new;

my @color = qw/red yellow pink green purple orange blue/;

my $listbox = $top->Scrolled('Listbox')->pack;

for (0..99){
    $listbox->insert('end',$_);
    $listbox->itemconfigure($_, -background => $color[rand(@color)]);
}

MainLoop;
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