There is an increasing need for legislation worldwide to monitor and assess the ecological status of marine ecosystems, due to increasing pressures from human activities. The costs and time of traditional analyses are high, while methods based on molecular analysis could reduce these costs and shorten evaluation times significantly. Some biotic indices used to assess the status need reference conditions to be applied. Hence, our objective here is to develop reference conditions for a molecular-based benthic index (M−gAMBI), which can be compared with a morphological one (M−AMBI). Using 6 years of data from estuaries and coasts, we have been able to set reference conditions for five water types, including richness, diversity and AMBI. However, one problem is the absence of the whole human pressure gradient in all water types, making difficult to validate the reference conditions. Based on the results of this research, the M−gAMBI index could be considered suitable for ecological status assessment since it meets most of the criteria for considering a genomics-based index suitable. However, it is considered necessary to advance in (i) improving the detection of errors in genomic methods and similarity with morphological methods, and (ii) an intercalibration exercise, allowing adjusting quality class boundaries and determining the ecological status in an equivalent manner using both methods.