This script ( from author unknown) works on a gif I tested.

#!/usr/bin/perl
#None of these is fast, mainly because of the overhead of calling Perl
#code for each pixel.
use strict;

my $imagefile = shift or die "No file specified\n";

sub rgba2hex {
    sprintf "%02x%02x%02x%02x", map { $_ || 0 } @_;
}

{
    use Imager;
    my %colors;
    my $img = Imager->new();
    $img->open( file => $imagefile ) or die $img->errstr;
    my ( $w, $h ) = ( $img->getwidth, $img->getheight );
    for my $i ( 0 .. $w - 1 ) {
        for my $j ( 0 .. $h - 1 ) {
            my $color = $img->getpixel( x => $i, y => $j );
            my $hcolor = rgba2hex $color->rgba();
            print "$hcolor ";
        $colors{$hcolor}++;
        }
    }

    printf "Imager: Number of colours: %d\n", scalar keys %colors;
}

{
    use GD;
    my %colors;
    my $gd = GD::Image->new($imagefile) or die "GD::Image->new($imagef
+ile)";
    my ( $w, $h ) = $gd->getBounds();
    for my $i ( 0 .. $w - 1 ) {
        for my $j ( 0 .. $h - 1 ) {
            my $index = $gd->getPixel( $i, $j );
            my $hcolor = rgba2hex( $gd->rgb($index), 0 );
            $colors{$hcolor}++;
        }
    }

    printf "GD: Number of colours: %d\n", scalar keys %colors;
}

{
    use Image::Magick;
    my %colors;
    my $img = Image::Magick->new();
    my $rc  = $img->Read($imagefile);
    die $rc if $rc;
    my ( $w, $h ) = $img->Get( 'width', 'height' );
    for my $i ( 0 .. $w - 1 ) {
        for my $j ( 0 .. $h - 1 ) {
            my $color = $img->Get("pixel[$i,$j]");
            my $hcolor = rgba2hex split /,/, $color;
            $colors{$hcolor}++;
        }
    }

    printf "Image::Magick: Number of colours: %d\n", scalar keys %colo
+rs;
}
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