small improvements: 
sub test_regexps2 {
    my ($seq1, $seq2, $window, $mismatch) = @_;
    my $retval = '';
    my %cache;
    my @mask = (0) x (length ($seq2) - $window);

    for my $start (0 .. (length ($seq1) - $window)) {
        my $part = substr ($seq1, $start, $window);

        $retval .= $cache{$part} ||= do {
            my $regex = build_regexp ($part, $mismatch);
            my @res = @mask;

            while ($seq2 =~ m/(?=$regex)/g) {
                $res[ pos $seq2 ] = 1;
            }
            join '', @res, "\n";
        };
    }

    $retval;
}

[download]
benchmarks for length 200, 400 and 600 
200:
                Rate  orig_poster   test_regexps test_regexps2
orig_poster   9.70/s            --          -51%          -81%
test_regexps  19.6/s          103%            --          -61%
test_regexps2 49.8/s          413%          153%            --

400:
                Rate  orig_poster   test_regexps test_regexps2
orig_poster   2.44/s            --          -52%          -84%
test_regexps  5.08/s          109%            --          -67%
test_regexps2 15.5/s          535%          205%            --

600:
                Rate  orig_poster   test_regexps test_regexps2
orig_poster   1.06/s            --          -54%          -86%
test_regexps  2.30/s          117%            --          -70%
test_regexps2 7.75/s          633%          237%            --

[download]

In reply to Re^2: Speed up DNA dotplot by happy.barney
in thread Speed up DNA dotplot by Microcebus

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